Nucleic acid encoding a wheat brittle-1 homolog

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding a brittle-1 homolog. The invention also relates to the construction of a chimeric gene encoding all or a portion of the brittle-1 homolog, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the brittle-1 homolog in a transformed host cell.

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 09/668,884, filed Sep. 25, 2000, now pending, which claimspriority benefit of the International Application No. PCT/US99/06583,filed Mar. 22, 1999, now pending, which claims priority benefit of U.S.Provisional Application No. 60/079,420, filed Mar. 26, 1998.

FIELD OF THE INVENTION

[0002] This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encodingcarbohydrate biosynthetic enzymes in plants and seeds.

BACKGROUND OF THE INVENTION

[0003] Brittle-1 is one of several corn genes that, when mutated, causethe accumulation of sugars, rather than starch, in developing cornseeds. It has been shown that the brittle-1 gene encodes a plastidicmembrane transporter that is involved in the transport of ADP-glucosefrom the cytosol to the plastid where it is used for starch biosynthesis(Shannon et al. (1998) Plant Physiol 117:1235-1252). In corn, the mutantphenotype suggests that inactivation of the brittle-1 gene causes areduction in starch accumulation. This reduction in starch accumulationpresumably causes an increase in concentration of various sugars whichin turn provides a large available pool of carbon for other metabolicpathways (Sullivan, T. D. et al. (1991) Plant Cell 3(12):1337-1348;Sullivan, T. D. et al. (1995) Planta 196(3):477-484). By manipulatingthe level of the brittle-1 protein in cells, it may be possible tomodulate the level (higher or lower) of starch accumulation in seeds,thereby controlling the level of undesirable carbohydrate concentration.

[0004] Callose or 1,3-beta-D-glucan synthesis is stimulated in responseto infection by a plant pathogen. Callose appears to act as a physicalbarrier against plant pathogens (Beffa, R. S. et al. (1996) Plant Cell8(6): 1001-1011). It has been recently shown that plant mutants that donot produce callose efficiently or mutants that degrade callose morerapidly than normal have increased risk of infection by specificpathogens (Beffa, R. S. et al. (1996) Plant Cell 8(6): 1001-1011). Theseobservations suggest that by modulating the level of callose in a plantcell it may be possible to manipulate plant host defense systems. Forexample, it may be possible to cause an increase in the production ofcallose and thus provide enhanced disease resistance. Furthermore, ahigh beta-glucan content in plant material is desirable becausebeta-glucan is a major component of soluble fiber. Soluble fiber isimportant in a healthy diet because in general soluble fiber has beenshown to reduce cholesterol levels in humans (Fastnaught, C. E. et al.(1996) Crop Science 36:941-946).

[0005] Few of the genes encoding these enzymes in barley, corn,soybeans, rice and wheat have been isolated and sequenced. For example,no barley, soybean or wheat genes have been reported for the brittle-1protein and no corn, rice, Vernonia or wheat genes have been reportedfor 1,3-beta-D-glucan synthase. Accordingly, the availability of nucleicacid sequences encoding all or a portion of these enzymes wouldfacilitate studies to better understand carbohydrate metabolism andfunction in plants, provide genetic tools for the manipulation of thesebiosynthetic pathways, and provide a means to control starch and1,3-beta-D-glucan synthesis in plant cells.

SUMMARY OF THE INVENTION

[0006] The present invention concerns an isolated polynucleotidecomprising: (a) a first nucleotide sequence encoding a first polypeptidecomprising at least 200 amino acids, wherein the amino acid sequence ofthe first polypeptide and the amino acid sequence of SEQ ID NO:4 or SEQID NO:10 have at least 70%, 80%, 85%, 90%, or 95% identity based on theClustal alignment method, (b) a second nucleotide sequence encoding asecond polypeptide comprising at least 200 amino acids, wherein theamino acid sequence of the second polypeptide and the amino acidsequence of SEQ ID NO:14 have at least 85%, 90%, or 95% identity basedon the Clustal alignment method, (c) a third nucleotide sequenceencoding a third polypeptide comprising at least 300 amino acids,wherein the amino acid sequence of the third polypeptide and the aminoacid sequence of SEQ ID NO:18 have at least 70%, 80%, 85%, 90%, or 95%identity based on the Clustal alignment method, or (d) the complement ofthe first, second, or third nucleotide sequence, wherein the complementand the first, second, or third nucleotide sequence contain the samenumber of nucleotides and are 100% complementary. The first polypeptidepreferably comprises the amino acid sequence of SEQ ID NO:4 or SEQ IDNO:10, the second polypeptide preferably comprises the amino acidsequence of SEQ ID NO:14, and the third polypeptide preferably comprisesthe amino acid sequence of SEQ ID NO:18. The first nucleotide sequencepreferably comprises the nucleotide sequence of SEQ ID NO:3 or SEQ IDNO:9, the second nucleotide sequence preferably comprises the nucleotidesequence of SEQ ID NO:13, and the third nucleotide sequence preferablycomprises the nucleotide sequence of SEQ ID NO:17. The first, second,and third polypeptides preferably are brittle-1 homologs.

[0007] In a second embodiment, the present invention relates to achimeric gene comprising any of the isolated polynucleotides of thepresent invention operably linked to a regulatory sequence, and a cell,a plant, and a seed comprising the chimeric gene.

[0008] In a third embodiment, the present invention relates to a vectorcomprising any of the isolated polynucleotides of the present invention.

[0009] In a fourth embodiment, the present invention relates to anisolated polynucleotide fragment comprising a nucleotide sequencecomprised by any of the polynucleotides of the present invention,wherein the nucleotide sequence contains at least 30, 40, or 60nucleotides.

[0010] In a fifth embodiment, the present invention concerns an isolatedpolypeptide comprising: (a) a first amino acid sequence comprising atleast 200 amino acids, wherein the first amino acid sequence and theamino acid sequence of SEQ ID NO:4 or SEQ ID NO:10 have at least 70%,80%, 85%, 90%, or 95% identity based on the Clustal alignment method,(b) a second amino acid sequence comprising at least 200 amino acids,wherein the second amino acid sequence and the amino acid sequence ofSEQ ID NO:14 have at least 85%, 90%, or 95% identity based on theClustal alignment method, or (c) a third amino acid sequence comprisingat least 300 amino acids, wherein the third amino acid sequence and theamino acid sequence of SEQ ID NO:18 have at least 70%, 80%, 85%, 90%, or95% identity based on the Clustal alignment method. The first amino acidsequence preferably comprises the amino acid sequence of SEQ ID NO:4 orSEQ ID NO:10, the second amino acid sequence preferably comprises theamino acid sequence of SEQ ID NO:14, and the third amino acid sequencepreferably comprises the amino acid sequence of SEQ ID NO:18. Thepolypeptide preferably is a brittle-1 homolog.

[0011] In a sixth embodiment, the present invention relates to a methodfor transforming a cell comprising transforming a cell with any of theisolated polynucleotides of the present invention, and the celltransformed by this method. Advantageously, the cell is eukaryotic,e.g., a yeast or plant cell, or prokaryotic, e.g., a bacterium.

[0012] In a seventh embodiment, the present invention relates to amethod for producing a transgenic plant comprising transforming a plantcell with any of the isolated polynucleotides of the present inventionand regenerating a plant from the transformed plant cell, the transgenicplant produced by this method, and the seed obtained from thistransgenic plant.

[0013] In an eighth embodiment, the present invention relates to avirus, preferably a baculovirus, comprising any of the isolatedpolynucleotides of the present invention or any of the chimeric genes ofthe present invention.

[0014] In a ninth embodiment, the invention relates to a method ofselecting an isolated polynucleotide that affects the level ofexpression of a brittle-1 homolog protein or enzyme activity in a hostcell, preferably a plant cell, the method comprising the steps of: (a)constructing an isolated polynucleotide of the present invention or anisolated chimeric gene of the present invention; (b) introducing theisolated polynucleotide or the isolated chimeric gene into a host cell;(c) measuring the level of the brittle-1 homolog protein or enzymeactivity in the host cell containing the isolated polynucleotide; and(d) comparing the level of the brittle-1 homolog protein or enzymeactivity in the host cell containing the isolated polynucleotide withthe level of the brittle-1 homolog protein or enzyme activity in thehost cell that does not contain the isolated polynucleotide.

[0015] In a tenth embodiment, the invention concerns a method ofobtaining a nucleic acid fragment encoding a substantial portion of abrittle-1 homolog protein, preferably a plant brittle-1 homolog protein,comprising the steps of: synthesizing an oligonucleotide primercomprising a nucleotide sequence of at least one of 60 (preferably atleast one of 40, most preferably at least one of 30) contiguousnucleotides derived from a nucleotide sequence selected from the groupconsisting of SEQ ID NOs:3, 9, 13, and 17, and the complement of suchnucleotide sequences; and amplifying a nucleic acid fragment (preferablya cDNA inserted in a cloning vector) using the oligonucleotide primer.The amplified nucleic acid fragment preferably will encode a substantialportion of a brittle-1 homolog protein amino acid sequence.

[0016] In an eleventh embodiment, this invention relates to a method ofobtaining a nucleic acid fragment encoding all or a substantial portionof the amino acid sequence encoding a brittle-1 homolog proteincomprising the steps of: probing a cDNA or genomic library with anisolated polynucleotide of the present invention; identifying a DNAclone that hybridizes with an isolated polynucleotide of the presentinvention; isolating the identified DNA clone; and sequencing the cDNAor genomic fragment that comprises the isolated DNA clone.

[0017] In a twelfth embodiment, this invention concerns a method forpositive selection of a transformed cell comprising: (a) transforming ahost cell with the chimeric gene of the present invention or anexpression cassette of the present invention; and (b) growing thetransformed host cell, preferably a plant cell, such as a monocot or adicot, under conditions which allow expression of the brittle-1 homologprotein polynucleotide in an amount sufficient to complement a nullmutant to provide a positive selection means.

[0018] In a thirteenth embodiment, this invention relates to a method ofaltering the level of expression of a brittle-1 homolog protein in ahost cell comprising: (a) transforming a host cell with a chimeric geneof the present invention; and (b) growing the transformed host cellunder conditions that are suitable for expression of the chimeric genewherein expression of the chimeric gene results in production of alteredlevels of the brittle-1 homolog protein in the transformed host cell.

BRIEF DESCRIPTION OF THE DRAWING AND SEQUENCE LISTINGS

[0019] The invention can be more fully understood from the followingdetailed description and the accompanying drawing and Sequence Listingwhich form a part of this application.

[0020]FIG. 1 depicts the amino acid sequence alignment between thebrittle-1 homologs encoded by the nucleotide sequences derived fromsoybean clone sfl1.pk0015.h4 (SEQ ID NO:10) and wheat clonewdk1c.pk012.c23 (SEQ ID NO:18), and Zea mays brittle-1 protein (NCBIGenBank Identifier (GI) No. 231654; SEQ ID NO:21). Amino acids which areconserved among all and at least two sequences with an amino acid atthat position are indicated with an asterisk (*). Dashes are used by theprogram to maximize alignment of the sequences.

[0021] Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. Table 1 also identifies the cDNA clonesas individual ESTs (“EST”), the sequences of the entire cDNA insertscomprising the indicated cDNA clones (“FIS”), contigs assembled from twoor more ESTs (“Contig”), contigs assembled from an FIS and one or moreESTs or PCR fragment sequence (“Contig*”), or sequences encoding theentire protein derived from an FIS, a contig, or an FIS and PCR fragmentsequence (“CGS”). SEQ ID NOs:1, 2, 5, 6, 7, 8, 11, 12, 15, 16, 19, and20 presented herein correspond to SEQ ID NOs:15, 16, 17, 18, 19, 20, 21,22, 25, 26, 27, and 28, respectively, presented in U.S. application Ser.No. 09/668,884, filed Sep. 25, 2000. The sequence descriptions andSequence Listing attached hereto comply with the rules governingnucleotide and/or amino acid sequence disclosures in patent applicationsas set forth in 37 C.F.R. §1.821-1.825. TABLE 1 Brittle-1 Homologs SEQID NO: Protein (Nucle- (Amino (Plant Source) Clone Designation Statusotide) Acid) Brittle-1 Homolog bsh1.pk0003.c5 EST 1 2 (Barley) Brittle-1Homolog bsh1.pk0003.c5 FIS 3 4 (Barley) Brittle-1 Homolog rsl1n.pk013.j2EST 5 6 (Rice) Brittle-1 Homolog sfl1.pk0015.h4 EST 7 8 (Soybean)Brittle-1 Homolog sfl1.pk0015.h4 (FIS) CGS 9 10 (Soybean) Brittle-1Homolog ssm.pk0058.al EST 11 12 (Soybean) Brittle-1 Homologssm.pk0058.al FIS 13 14 (Soybean) Brittle-1 Homolog wdk1c.pk012.c23 EST15 16 (Wheat) Brittle-1 Homolog wdk1c.pk012.c23 CGS 17 18 (Wheat) (FIS)Brittle-1 Homolog wre1n.pk0049.e1 FIS 19 20 (Wheat)

[0022] The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J 219 (No.2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

[0023] In the context of this disclosure, a number of terms shall beutilized. The terms “polynucleotide”, “polynucleotide sequence”,“nucleic acid sequence”, and “nucleic acid fragment”/“isolated nucleicacid fragment” are used interchangeably herein. These terms encompassnucleotide sequences and the like. A polynucleotide may be a polymer ofRNA or DNA that is single- or double-stranded, that optionally containssynthetic, non-natural or altered nucleotide bases. A polynucleotide inthe form of a polymer of DNA may be comprised of one or more segments ofcDNA, genomic DNA, synthetic DNA, or mixtures thereof. An isolatedpolynucleotide of the present invention may include at least 60contiguous nucleotides, preferably at least 40 contiguous nucleotides,most preferably at least 30 contiguous nucleotides derived from SEQ IDNOs:3, 9, 13, or 17, or the complement of such sequences.

[0024] The term “brittle-1 homolog” refers to a protein whose amino acidsequence has at least 50%, 60%, 70%, 80%, 85%, 90% or 95% identity withSEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:14, or SEQ ID NO:18 based on theClustal alignment method using default parameters, and has brittle-1activity (i.e., as adenylate translocator) as described in Shannon etal. (1998) Plant Physiol 117:1235-1252.

[0025] The term “isolated” polynucleotide refers to a polynucleotidethat is substantially free from other nucleic acid sequences, such asand not limited to other chromosomal and extrachromosomal DNA and RNA.Isolated polynucleotides may be purified from a host cell in which theynaturally occur. Conventional nucleic acid purification methods known toskilled artisans may be used to obtain isolated polynucleotides. Theterm also embraces recombinant polynucleotides and chemicallysynthesized polynucleotides.

[0026] The term “recombinant” means, for example, that a nucleic acidsequence is made by an artificial combination of two otherwise separatedsegments of sequence, e.g., by chemical synthesis or by the manipulationof isolated nucleic acids by genetic engineering techniques.

[0027] As used herein, “contig” refers to a nucleotide sequence that isassembled from two or more constituent nucleotide sequences that sharecommon or overlapping regions of sequence homology. For example, thenucleotide sequences of two or more nucleic acid fragments can becompared and aligned in order to identify common or overlappingsequences. Where common or overlapping sequences exist between two ormore nucleic acid fragments, the sequences (and thus their correspondingnucleic acid fragments) can be assembled into a single contiguousnucleotide sequence.

[0028] As used herein, “substantially similar” refers to nucleic acidfragments wherein changes in one or more nucleotide bases results insubstitution of one or more amino acids, but do not affect thefunctional properties of the polypeptide encoded by the nucleotidesequence. “Substantially similar” also refers to nucleic acid fragmentswherein changes in one or more nucleotide bases does not affect theability of the nucleic acid fragment to mediate alteration of geneexpression by gene silencing through for example antisense orco-suppression technology. “Substantially similar” also refers tomodifications of the nucleic acid fragments of the instant inventionsuch as deletion or insertion of one or more nucleotides that do notsubstantially affect the functional properties of the resultingtranscript vis-à-vis the ability to mediate gene silencing or alterationof the functional properties of the resulting protein molecule. It istherefore understood that the invention encompasses more than thespecific exemplary nucleotide or amino acid sequences and includesfunctional equivalents thereof. The terms “substantially similar” and“corresponding substantially” are used interchangeably herein.

[0029] Substantially similar nucleic acid fragments may be selected byscreening nucleic acid fragments representing subfragments ormodifications of the nucleic acid fragments of the instant invention,wherein one or more nucleotides are substituted, deleted and/orinserted, for their ability to affect the level of the polypeptideencoded by the unmodified nucleic acid fragment in a plant or plantcell. For example, a substantially similar nucleic acid fragmentrepresenting at least 30 contiguous nucleotides derived from the instantnucleic acid fragment can be constructed and introduced into a plant orplant cell. The level of the polypeptide encoded by the unmodifiednucleic acid fragment present in a plant or plant cell exposed to thesubstantially similar nucleic fragment can then be compared to the levelof the polypeptide in a plant or plant cell that is not exposed to thesubstantially similar nucleic acid fragment.

[0030] For example, it is well known in the art that antisensesuppression and co-suppression of gene expression may be accomplishedusing nucleic acid fragments representing less than the entire codingregion of a gene, and by using nucleic acid fragments that do not share100% sequence identity with the gene to be suppressed. Moreover,alterations in a nucleic acid fragment which result in the production ofa chemically equivalent amino acid at a given site, but do not effectthe functional properties of the encoded polypeptide, are well known inthe art. Thus, a codon for the amino acid alanine, a hydrophobic aminoacid, may be substituted by a codon encoding another less hydrophobicresidue, such as glycine, or a more hydrophobic residue, such as valine,leucine, or isoleucine. Similarly, changes which result in substitutionof one negatively charged residue for another, such as aspartic acid forglutamic acid, or one positively charged residue for another, such aslysine for arginine, can also be expected to produce a functionallyequivalent product. Nucleotide changes which result in alteration of theN-terminal and C-terminal portions of the polypeptide molecule wouldalso not be expected to alter the activity of the polypeptide. Each ofthe proposed modifications is well within the routine skill in the art,as is determination of retention of biological activity of the encodedproducts. Consequently, an isolated polynucleotide comprising anucleotide sequence of at least 60 (preferably at least 40, mostpreferably at least 30) contiguous nucleotides derived from a nucleotidesequence selected from the group consisting of SEQ ID NOs:3, 9, 13, and17, and the complement of such nucleotide sequences may be used inmethods of selecting an isolated polynucleotide that affects theexpression of a brittle-1 homolog polypeptide in a host cell. A methodof selecting an isolated polynucleotide that affects the level ofexpression of a polypeptide in a virus or in a host cell (eukaryotic,such as plant or yeast, prokaryotic such as bacterial) may comprise thesteps of: constructing an isolated polynucleotide of the presentinvention or an isolated chimeric gene of the present invention;introducing the isolated polynucleotide or the isolated chimeric geneinto a host cell; measuring the level of a polypeptide or enzymeactivity in the host cell containing the isolated polynucleotide; andcomparing the level of a polypeptide or enzyme activity in the host cellcontaining the isolated polynucleotide with the level of a polypeptideor enzyme activity in a host cell that does not contain the isolatedpolynucleotide.

[0031] Moreover, substantially similar nucleic acid fragments may alsobe characterized by their ability to hybridize. Estimates of suchhomology are provided by either DNA-DNA or DNA-RNA hybridization underconditions of stringency as is well understood by those skilled in theart (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRLPress, Oxford, U.K.). Stringency conditions can be adjusted to screenfor moderately similar fragments, such as homologous sequences fromdistantly related organisms, to highly similar fragments, such as genesthat duplicate functional enzymes from closely related organisms.Post-hybridization washes determine stringency conditions. One set ofpreferred conditions uses a series of washes starting with 6×SSC, 0.5%SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDSat 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at50° C. for 30 min. A more preferred set of stringent conditions useshigher temperatures in which the washes are identical to those aboveexcept for the temperature of the final two 30 min washes in 0.2×SSC,0.5% SDS was increased to 60° C. Another preferred set of highlystringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65°C.

[0032] Substantially similar nucleic acid fragments of the instantinvention may also be characterized by the percent identity of the aminoacid sequences that they encode to the amino acid sequences disclosedherein, as determined by algorithms commonly employed by those skilledin this art. Suitable nucleic acid fragments (isolated polynucleotidesof the present invention) encode polypeptides that are at least about70% identical, preferably at least about 80% identical to the amino acidsequences reported herein. Preferred nucleic acid fragments encode aminoacid sequences that are at least about 85% identical to the amino acidsequences reported herein. More preferred nucleic acid fragments encodeamino acid sequences that are at least about 90% identical to the aminoacid sequences reported herein. Most preferred are nucleic acidfragments that encode amino acid sequences that are at least about 95%identical to the amino acid sequences reported herein. Suitable nucleicacid fragments not only have the above identities but typically encode apolypeptide having at least 50 amino acids, preferably at least 100amino acids, more preferably at least 150 amino acids, still morepreferably at least 200 amino acids, and most preferably at least 250 or300 amino acids. Sequence alignments and percent identity calculationswere performed using the Megalign program of the LASERGENEbioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiplealignment of the sequences was performed using the Clustal method ofalignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the defaultparameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parametersfor pairwise alignments using the Clustal method were KTUPLE 1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0033] A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more contiguous nucleotides isnecessary in order to putatively identify a polypeptide or nucleic acidsequence as homologous to a known protein or gene. Moreover, withrespect to nucleotide sequences, gene-specific oligonucleotide probescomprising 30 or more contiguous nucleotides may be used insequence-dependent methods of gene identification (e.g., Southernhybridization) and isolation (e.g., in situ hybridization of bacterialcolonies or bacteriophage plaques). In addition, short oligonucleotidesof 12 or more nucleotides may be used as amplification primers in PCR inorder to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises a nucleotide sequence that will afford specific identificationand/or isolation of a nucleic acid fragment comprising the sequence. Theinstant specification teaches amino acid and nucleotide sequencesencoding polypeptides that comprise one or more particular plantproteins. The skilled artisan, having the benefit of the sequences asreported herein, may now use all or a substantial portion of thedisclosed sequences for purposes known to those skilled in this art.Accordingly, the instant invention comprises the complete sequences asreported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

[0034] “Codon degeneracy” refers to divergence in the genetic codepermitting variation of the nucleotide sequence without effecting theamino acid sequence of an encoded polypeptide. Accordingly, the instantinvention relates to any nucleic acid fragment comprising a nucleotidesequence that encodes all or a substantial portion of the amino acidsequences set forth herein. The skilled artisan is well aware of the“codon-bias” exhibited by a specific host cell in usage of nucleotidecodons to specify a given amino acid. Therefore, when synthesizing anucleic acid fragment for improved expression in a host cell, it isdesirable to design the nucleic acid fragment such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

[0035] “Synthetic nucleic acid fragments” can be assembled fromoligonucleotide building blocks that are chemically synthesized usingprocedures known to those skilled in the art. These building blocks areligated and annealed to form larger nucleic acid fragments which maythen be enzymatically assembled to construct the entire desired nucleicacid fragment. “Chemically synthesized”, as related to a nucleic acidfragment, means that the component nucleotides were assembled in vitro.Manual chemical synthesis of nucleic acid fragments may be accomplishedusing well established procedures, or automated chemical synthesis canbe performed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of the nucleotide sequence to reflectthe codon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

[0036] “Gene” refers to a nucleic acid fragment that expresses aspecific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence. “Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign-gene” refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

[0037] “Coding sequence” refers to a nucleotide sequence that codes fora specific amino acid sequence. “Regulatory sequences” refer tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

[0038] “Promoter” refers to a nucleotide sequence capable of controllingthe expression of a coding sequence or functional RNA. In general, acoding sequence is located 3′ to a promoter sequence. The promotersequence consists of proximal and more distal upstream elements, thelatter elements often referred to as enhancers. Accordingly, an“enhancer” is a nucleotide sequence which can stimulate promoteractivity and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue-specificity of apromoter. Promoters may be derived in their entirety from a native gene,or may be composed of different elements derived from differentpromoters found in nature, or may even comprise synthetic nucleotidesegments. It is understood by those skilled in the art that differentpromoters may direct the expression of a gene in different tissues orcell types, or at different stages of development, or in response todifferent environmental conditions. Promoters which cause a nucleic acidfragment to be expressed in most cell types at most times are commonlyreferred to as “constitutive promoters”. New promoters of various typesuseful in plant cells are constantly being discovered; numerous examplesmay be found in the compilation by Okamuro and Goldberg (1989)Biochemistry of Plants 15:1-82. It is further recognized that since inmost cases the exact boundaries of regulatory sequences have not beencompletely defined, nucleic acid fragments of different lengths may haveidentical promoter activity.

[0039] “Translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) Mol. Biotechnol.3:225-236).

[0040] “3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

[0041] “RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptides by the cell. “cDNA” refers to DNA that is complementary toand derived from an mRNA template. The cDNA can be single-stranded orconverted to double stranded form using, for example, the Klenowfragment of DNA polymerase I. “Sense-RNA” refers to an RNA transcriptthat includes the mRNA and so can be translated into a polypeptide bythe cell. “Antisense RNA” refers to an RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

[0042] The term “operably linked” refers to the association of two ormore nucleic acid fragments on a single polynucleotide so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

[0043] The term “expression”, as used herein, refers to thetranscription and stable accumulation of sense (mRNA) or antisense RNAderived from the nucleic acid fragment of the invention. Expression mayalso refer to translation of mRNA into a polypeptide. “Antisenseinhibition” refers to the production of antisense RNA transcriptscapable of suppressing the expression of the target protein.“Overexpression” refers to the production of a gene product intransgenic organisms that exceeds levels of production in normal ornon-transformed organisms. “Co-suppression” refers to the production ofsense RNA transcripts capable of suppressing the expression of identicalor substantially similar foreign or endogenous genes (U.S. Pat. No.5,231,020, incorporated herein by reference).

[0044] A “protein” or “polypeptide” is a chain of amino acids arrangedin a specific order determined by the coding sequence in apolynucleotide encoding the polypeptide. Each protein or polypeptide hasa unique function.

[0045] “Altered levels” or “altered expression” refers to the productionof gene product(s) in transgenic organisms in amounts or proportionsthat differ from that of normal or non-transformed organisms.

[0046] “Null mutant” refers here to a host cell which either lacks theexpression of a certain polypeptide or expresses a polypeptide which isinactive or does not have any detectable expected enzymatic function.

[0047] “Mature protein” or the term “mature” when used in describing aprotein refers to a post-translationally processed polypeptide; i.e.,one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor protein” or the term“precursor” when used in describing a protein refers to the primaryproduct of translation of mRNA; i.e., with pre- and propeptides stillpresent. Pre- and propeptides may be but are not limited tointracellular localization signals.

[0048] A “chloroplast transit peptide” is an amino acid sequence whichis translated in conjunction with a protein and directs the protein tothe chloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100:1627-1632).

[0049] “Transformation” refers to the transfer of a nucleic acidfragment into the genome of a host organism, resulting in geneticallystable inheritance. Host organisms containing the transformed nucleicacid fragments are referred to as “transgenic” organisms. Examples ofmethods of plant transformation include Agrobacterium-mediatedtransformation (De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference). Thus, isolated polynucleotides of thepresent invention can be incorporated into recombinant constructs,typically DNA constructs, capable of introduction into and replicationin a host cell. Such a construct can be a vector that includes areplication system and sequences that are capable of transcription andtranslation of a polypeptide-encoding sequence in a given host cell. Anumber of vectors suitable for stable transfection of plant cells or forthe establishment of transgenic plants have been described in, e.g.,Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987;Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989; and Flevin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990. Typically, plant expression vectors include,for example, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

[0050] Standard recombinant DNA and molecular cloning techniques usedherein are well known in the art and are described more fully inSambrook et al. Molecular Cloning: A Laboratory Manual; Cold SpringHarbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter“Maniatis”).

[0051] “PCR” or “polymerase chain reaction” is well known by thoseskilled in the art as a technique used for the amplification of specificDNA segments (U.S. Pat. Nos. 4,683,195 and 4,800,159).

[0052] The present invention concerns an isolated polynucleotidecomprising: (a) a first nucleotide sequence encoding a first polypeptidecomprising at least 200 amino acids, wherein the amino acid sequence ofthe first polypeptide and the amino acid sequence of SEQ ID NO:4 or SEQID NO:10 have at least 70%, 80%, 85%, 90%, or 95% identity based on theClustal alignment method, (b) a second nucleotide sequence encoding asecond polypeptide comprising at least 200 amino acids, wherein theamino acid sequence of the second polypeptide and the amino acidsequence of SEQ ID NO:14 have at least 85%, 90%, or 95% identity basedon the Clustal alignment method, (c) a third nucleotide sequenceencoding a third polypeptide comprising at least 300 amino acids,wherein the amino acid sequence of the third polypeptide and the aminoacid sequence of SEQ ID NO:18 have at least 70%, 80%, 85%, 90%, or 95%identity based on the Clustal alignment method, or (d) the complement ofthe first, second, or third nucleotide sequence, wherein the complementand the first, second, or third nucleotide sequence contain the samenumber of nucleotides and are 100% complementary. The first polypeptidepreferably comprises the amino acid sequence of SEQ ID NO:4 or SEQ IDNO:10, the second polypeptide preferably comprises the amino acidsequence of SEQ ID NO:14, and the third polypeptide preferably comprisesthe amino acid sequence of SEQ ID NO:18. The first nucleotide sequencepreferably comprises the nucleotide sequence of SEQ ID NO:3 or SEQ IDNO:9, the second nucleotide sequence preferably comprises the nucleotidesequence of SEQ ID NO:13, and the third nucleotide sequence preferablycomprises the nucleotide sequence of SEQ ID NO:17. The first, second,and third polypeptides preferably are brittle-1 homologs.

[0053] Nucleic acid fragments encoding at least a portion of severalbrittle-1 homologs have been isolated and identified by comparison ofrandom plant cDNA sequences to public databases containing nucleotideand protein sequences using the BLAST algorithms well known to thoseskilled in the art. The nucleic acid fragments of the instant inventionmay be used to isolate cDNAs and genes encoding homologous proteins fromthe same or other plant species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g., polymerase chain reaction, ligase chain reaction).

[0054] For example, genes encoding other brittle-1 homologs, either ascDNAs or genomic DNAs, could be isolated directly by using all or aportion of the instant nucleic acid fragments as DNA hybridizationprobes to screen libraries from any desired plant employing methodologywell known to those skilled in the art. Specific oligonucleotide probesbased upon the instant nucleic acid sequences can be designed andsynthesized by methods known in the art (Maniatis). Moreover, an entiresequence can be used directly to synthesize DNA probes by methods knownto the skilled artisan such as random primer DNA labeling, nicktranslation, end-labeling techniques, or RNA probes using available invitro transcription systems. In addition, specific primers can bedesigned and used to amplify a part or all of the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full length cDNA or genomic fragments underconditions of appropriate stringency.

[0055] In addition, two short segments of the instant nucleic acidfragments may be used in polymerase chain reaction protocols to amplifylonger nucleic acid fragments encoding homologous genes from DNA or RNA.The polymerase chain reaction may also be performed on a library ofcloned nucleic acid fragments wherein the sequence of one primer isderived from the instant nucleic acid fragments, and the sequence of theother primer takes advantage of the presence of the polyadenylic acidtracts to the 3′ end of the mRNA precursor encoding plant genes.Alternatively, the second primer sequence may be based upon sequencesderived from the cloning vector. For example, the skilled artisan canfollow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci.USA 85:8998-9002) to generate cDNAs by using PCR to amplify copies ofthe region between a single point in the transcript and the 3′ or 5′end. Primers oriented in the 3′ and 5′ directions can be designed fromthe instant sequences. Using commercially available 3′ RACE or 5′ RACEsystems (BRL), specific 3′ or 5′ cDNA fragments can be isolated (Oharaet al. (1989) Proc. Natl. Acad. Sci. USA 86:5673-5677; Loh et al. (1989)Science 243:217-220). Products generated by the 3′ and 5′ RACEprocedures can be combined to generate full-length cDNAs (Frohman andMartin (1989) Techniques 1:165). Consequently, a polynucleotidecomprising a nucleotide sequence of at least 60 (preferably at least 40,most preferably at least 30) contiguous nucleotides derived from anucleotide sequence selected from the group consisting of SEQ ID NOs:3,9, 13, and 17 and the complement of such nucleotide sequences may beused in such methods to obtain a nucleic acid fragment encoding asubstantial portion of an amino acid sequence of a polypeptide.

[0056] The present invention relates to a method of obtaining a nucleicacid fragment encoding a substantial portion of a brittle-1 homologpolypeptide, preferably a substantial portion of a plant brittle-1homolog polypeptide, comprising the steps of: synthesizing anoligonucleotide primer comprising a nucleotide sequence of at least 60(preferably at least 40, most preferably at least 30) contiguousnucleotides derived from a nucleotide sequence selected from the groupconsisting of SEQ ID NOs:3, 9, 13, and 17, and the complement of suchnucleotide sequences; and amplifying a nucleic acid fragment (preferablya cDNA inserted in a cloning vector) using the oligonucleotide primer.The amplified nucleic acid fragment preferably will encode a portion ofa brittle-1 homolog polypeptide.

[0057] Availability of the instant nucleotide and deduced amino acidsequences facilitates immunological screening of cDNA expressionlibraries. Synthetic peptides representing portions of the instant aminoacid sequences may be synthesized. These peptides can be used toimmunize animals to produce polyclonal or monoclonal antibodies withspecificity for peptides or proteins comprising the amino acidsequences. These antibodies can be then be used to screen cDNAexpression libraries to isolate full-length cDNA clones of interest(Lemer (1984) Adv. Immunol. 36:1-34; Maniatis).

[0058] In another embodiment, this invention concerns viruses and hostcells comprising either the chimeric genes of the invention as describedherein or an isolated polynucleotide of the invention as describedherein. Examples of host cells which can be used to practice theinvention include, but are not limited to, yeast, bacteria, and plants.

[0059] As was noted above, the nucleic acid fragments of the instantinvention may be used to create transgenic plants in which the disclosedpolypeptides are present at higher or lower levels than normal or incell types or developmental stages in which they are not normally found.This would have the effect of altering the level of starch synthesis andaccumulation in those cells.

[0060] Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.The chimeric gene may comprise promoter sequences and translation leadersequences derived from the same genes. 3′ Non-coding sequences encodingtranscription termination signals may also be provided. The instantchimeric gene may also comprise one or more introns in order tofacilitate gene expression.

[0061] Plasmid vectors comprising the instant isolated polynucleotide(or chimeric gene) may be constructed. The choice of plasmid vector isdependent upon the method that will be used to transform host plants.The skilled artisan is well aware of the genetic elements that must bepresent on the plasmid vector in order to successfully transform, selectand propagate host cells containing the chimeric gene. The skilledartisan will also recognize that different independent transformationevents will result in different levels and patterns of expression (Joneset al. (1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen.Genetics 218:78-86), and thus that multiple events must be screened inorder to obtain lines displaying the desired expression level andpattern. Such screening may be accomplished by Southern analysis of DNA,Northern analysis of mRNA expression, Western analysis of proteinexpression, or phenotypic analysis.

[0062] For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by directing the codingsequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel (1992) Plant Phys. 100:1627-1632) with or without removingtargeting sequences that are already present. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of use may be discovered in the future.

[0063] It may also be desirable to reduce or eliminate expression ofgenes encoding the instant polypeptides in plants for some applications.In order to accomplish this, a chimeric gene designed for co-suppressionof the instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

[0064] Molecular genetic solutions to the generation of plants withaltered gene expression have a decided advantage over more traditionalplant breeding approaches. Changes in plant phenotypes can be producedby specifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression of aspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

[0065] The person skilled in the art will know that specialconsiderations are associated with the use of antisense or cosuppressiontechnologies in order to reduce expression of particular genes. Forexample, the proper level of expression of sense or antisense genes mayrequire the use of different chimeric genes utilizing differentregulatory elements known to the skilled artisan. Once transgenic plantsare obtained by one of the methods described above, it will be necessaryto screen individual transgenics for those that most effectively displaythe desired phenotype. Accordingly, the skilled artisan will developmethods for screening large numbers of transformants. The nature ofthese screens will generally be chosen on practical grounds. Forexample, one can screen by looking for changes in gene expression byusing antibodies specific for the protein encoded by the gene beingsuppressed, or one could establish assays that specifically measureenzyme activity. A preferred method will be one which allows largenumbers of samples to be processed rapidly, since it will be expectedthat a large number of transformants will be negative for the desiredphenotype.

[0066] In another embodiment, the present invention concerns an isolatedpolypeptide comprising: (a) a first amino acid sequence comprising atleast 200 amino acids, wherein the first amino acid sequence and theamino acid sequence of SEQ ID NO:4 or SEQ ID NO:10 have at least 70%,80%, 85%, 90%, or 95% identity based on the Clustal alignment method,(b) a second amino acid sequence comprising at least 200 amino acids,wherein the second amino acid sequence and the amino acid sequence ofSEQ ID NO:14 have at least 85%, 90%, or 95% identity based on theClustal alignment method, or (c) a third amino acid sequence comprisingat least 300 amino acids, wherein the third amino acid sequence and theamino acid sequence of SEQ ID NO:18 have at least 70%, 80%, 85%, 90%, or95% identity based on the Clustal alignment method. The first amino acidsequence preferably comprises the amino acid sequence of SEQ ID NO:4 orSEQ ID NO:10, the second amino acid sequence preferably comprises theamino acid sequence of SEQ ID NO:14, and the third amino acid sequencepreferably comprises the amino acid sequence of SEQ ID NO:18. Thepolypeptide preferably is a brittle-1 homolog.

[0067] The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to these proteins by methods wellknown to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a chimeric gene for production of the instant polypeptides.This chimeric gene could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe encoded brittle-1 homolog. An example of a vector for high levelexpression of the instant polypeptides in a bacterial host is provided(Example 6).

[0068] All or a substantial portion of the polynucleotides of theinstant invention may also be used as probes for genetically andphysically mapping the genes that they are a part of, and used asmarkers for traits linked to those genes. Such information may be usefulin plant breeding in order to develop lines with desired phenotypes. Forexample, the instant nucleic acid fragments may be used as restrictionfragment length polymorphism (RFLP) markers. Southern blots (Maniatis)of restriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet.32:314-331).

[0069] The production and use of plant gene-derived probes for use ingenetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol.Biol. Reporter 4.37-41. Numerous publications describe genetic mappingof specific cDNA clones using the methodology outlined above orvariations thereof. For example, F2 intercross populations, backcrosspopulations, randomly mated populations, near isogenic lines, and othersets of individuals may be used for mapping. Such methodologies are wellknown to those skilled in the art.

[0070] Nucleic acid probes derived from the instant nucleic acidsequences may also be used for physical mapping (i.e., placement ofsequences on physical maps; see Hoheisel et al. In: Nonmammalian GenomicAnalysis: A Practical Guide, Academic press 1996, pp. 319-346, andreferences cited therein).

[0071] In another embodiment, nucleic acid probes derived from theinstant nucleic acid sequences may be used in direct fluorescence insitu hybridization (FISH) mapping (Trask (1991) Trends Genet.7:149-154). Although current methods of FISH mapping favor use of largeclones (several to several hundred KB; see Laan et al. (1995) GenomeRes. 5:13-20), improvements in sensitivity may allow performance of FISHmapping using shorter probes.

[0072] A variety of nucleic acid amplification-based methods of geneticand physical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat.Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic AcidRes. 17:6795-6807). For these methods, the sequence of a nucleic acidfragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

[0073] Loss of function mutant phenotypes may be identified for theinstant cDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci USA 86:9402-9406; Koes et al. (1995)Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell7:75-84). The latter approach may be accomplished in two ways. First,short segments of the instant nucleic acid fragments may be used inpolymerase chain reaction protocols in conjunction with a mutation tagsequence primer on DNAs prepared from a population of plants in whichMutator transposons or some other mutation-causing DNA element has beenintroduced (see Bensen, supra). The amplification of a specific DNAfragment with these primers indicates the insertion of the mutation tagelement in or near the plant gene encoding the instant polypeptide.Alternatively, the instant nucleic acid fragment may be used as ahybridization probe against PCR amplification products generated fromthe mutation population using the mutation tag sequence primer inconjunction with an arbitrary genomic site primer, such as that for arestriction enzyme site-anchored synthetic adaptor. With either method,a plant containing a mutation in the endogenous gene encoding theinstant polypeptide can be identified and obtained. This mutant plantcan then be used to determine or confirm the natural function of theinstant polypeptides disclosed herein.

EXAMPLES

[0074] The present invention is further defined in the followingExamples, in which parts and percentages are by weight and degrees areCelsius, unless otherwise stated. It should be understood that theseExamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theinvention to adapt it to various usages and conditions. Thus, variousmodifications of the invention in addition to those shown and describedherein will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims.

[0075] The disclosure of each reference set forth herein is incorporatedherein by reference in its entirety.

Example 1 Composition of cDNA Libraries; Isolation and Sequencing ofcDNA Clones

[0076] cDNA libraries representing mRNAs from various barley (Hordeumvulgare), rice (Oryza sativa), soybean (Glycine max), and wheat(Triticum aestivum) tissues were prepared. The characteristics of thelibraries are described below. TABLE 2 eDNA Libraries from Barley, Rice,Soybean, and Wheat Library Tissue Clone bsh1 Barley Sheath, DevelopingSeedling bsh1.pk0003.c5 rsl1n Rice 15-Day-Old Seedling* rsl1n.pk013.j2sfl1 Soybean Immature Flower sfl1.pk0015.h4 ssm Soybean Shoot Meristemssm.pk0058.a1 wdk1c Wheat Developing Kernel, 3 Days Afterwdk1c.pk012.c23 Anthesis wre1n Wheat Root From 7 Day Old Etiolatedwre1n.pk0049.e1 Seedling*

[0077] cDNA libraries may be prepared by any one of many methodsavailable. For example, the cDNAs may be introduced into plasmid vectorsby first preparing the cDNA libraries in Uni-ZAP™ XR vectors accordingto the manufacturer's protocol (Stratagene Cloning Systems, La Jolla,Calif.). The Uni-ZAP™ XR libraries are converted into plasmid librariesaccording to the protocol provided by Stratagene. Upon conversion, cDNAinserts will be contained in the plasmid vector pBluescript. Inaddition, the cDNAs may be introduced directly into precut Bluescript IISK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs),followed by transfection into DH10B cells according to themanufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts arein plasmid vectors, plasmid DNAs are prepared from randomly pickedbacterial colonies containing recombinant pBluescript plasmids, or theinsert cDNA sequences are amplified via polymerase chain reaction usingprimers specific for vector sequences flanking the inserted cDNAsequences. Amplified insert DNAs or plasmid DNAs are sequenced indye-primer sequencing reactions to generate partial cDNA sequences(expressed sequence tags or “ESTs”; see Adams et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

[0078] Full-insert sequence (FIS) data is generated utilizing a modifiedtransposition protocol. Clones identified for FIS are recovered fromarchived glycerol stocks as single colonies, and plasmid DNAs areisolated via alkaline lysis. Isolated DNA templates are reacted withvector primed M13 forward and reverse oligonucleotides in a PCR-basedsequencing reaction and loaded onto automated sequencers. Confirmationof clone identification is performed by sequence alignment to theoriginal EST sequence from which the FIS request is made.

[0079] Confirmed templates are transposed via the Primer Islandtransposition kit (PE Applied Biosystems, Foster City, Calif.) which isbased upon the Saccharomyces cerevisiae Ty1 transposable element (Devineand Boeke (1994) Nucleic Acids Res. 22:3765-3772). The in vitrotransposition system places unique binding sites randomly throughout apopulation of large DNA molecules. The transposed DNA is then used totransform DH10B electro-competent cells (Gibco BRL/Life Technologies,Rockville, Md.) via electroporation. The transposable element containsan additional selectable marker (named DHFR; Fling and Richards (1983)Nucleic Acids Res. 11:5147-5158), allowing for dual selection on agarplates of only those subclones containing the integrated transposon.Multiple subclones are randomly selected from each transpositionreaction, plasmid DNAs are prepared via alkaline lysis, and templatesare sequenced (ABI Prism dye-terminator ReadyReaction mix) outward fromthe transposition event site, utilizing unique primers specific to thebinding sites within the transposon.

[0080] Sequence data is collected (ABI Prism Collections) and assembledusing Phred/Phrap (P. Green, University of Washington, Seattle).Phrep/Phrap is a public domain software program which re-reads the ABIsequence data, re-calls the bases, assigns quality values, and writesthe base calls and quality values into editable output files. The Phrapsequence assembly program uses these quality values to increase theaccuracy of the assembled sequence contigs. Assemblies are viewed by theConsed sequence editor (D. Gordon, University of Washington, Seattle).

[0081] In some of the clones the cDNA fragment corresponds to a portionof the 3′-terminus of the gene and does not cover the entire openreading frame. In order to obtain the upstream information one of twodifferent protocols are used. The first of these methods results in theproduction of a fragment of DNA containing a portion of the desired genesequence while the second method results in the production of a fragmentcontaining the entire open reading frame. Both of these methods use tworounds of PCR amplification to obtain fragments from one or morelibraries. The libraries some times are chosen based on previousknowledge that the specific gene should be found in a certain tissue andsome times are randomly-chosen. Reactions to obtain the same gene may beperformed on several libraries in parallel or on a pool of libraries.Library pools are normally prepared using from 3 to 5 differentlibraries and normalized to a uniform dilution. In the first round ofamplification both methods use a vector-specific (forward) primercorresponding to a portion of the vector located at the 5′-terminus ofthe clone coupled with a gene-specific (reverse) primer. The firstmethod uses a sequence that is complementary to a portion of the alreadyknown gene sequence while the second method uses a gene-specific primercomplementary to a portion of the 3′-untranslated region (also referredto as UTR). In the second round of amplification a nested set of primersis used for both methods. The resulting DNA fragment is ligated into apBluescript vector using a commercial kit and following themanufacturer's protocol. This kit is selected from many available fromseveral vendors including Invitrogen (Carlsbad, Calif.), Promega Biotech(Madison, Wis.), and Gibco-BRL (Gaithersburg, Md.). The plasmid DNA isisolated by alkaline lysis method and submitted for sequencing andassembly using Phred/Phrap, as above.

Example 2 Identification of cDNA Clones

[0082] cDNA clones encoding brittle-1 homologs were identified byconducting BLAST (Basic Local Alignment Search Tool; Altschul et al.(1993) J. Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/)searches for similarity to sequences contained in the BLAST “nr”database (comprising all non-redundant GenBank CDS translations,sequences derived from the 3-dimensional structure Brookhaven ProteinData Bank, the last major release of the SWISS-PROT protein sequencedatabase, EMBL, and DDBJ databases). The cDNA sequences obtained inExample 1 were analyzed for similarity to all publicly available DNAsequences contained in the “nr” database using the BLASTN algorithmprovided by the National Center for Biotechnology Information (NCBI).The DNA sequences were translated in all reading frames and compared forsimilarity to all publicly available protein sequences contained in the“nr” database using the BLASTX algorithm (Gish and States (1993) Nat.Genet. 3:266-272) provided by the NCBI. For convenience, the P-value(probability) of observing a match of a cDNA sequence to a sequencecontained in the searched databases merely by chance as calculated byBLAST are reported herein as “pLog” values, which represent the negativeof the logarithm of the reported P-value. Accordingly, the greater thepLog value, the greater the likelihood that the cDNA sequence and theBLAST “hit” represent homologous proteins.

[0083] ESTs submitted for analysis are compared to the genbank databaseas described above. ESTs that contain sequences more 5- or 3-prime canbe found by using the BLASTn algorithm (Altschul et al (1997) NucleicAcids Res. 25:3389-3402.) against the DuPont proprietary databasecomparing nucleotide sequences that share common or overlapping regionsof sequence homology. Where common or overlapping sequences existbetween two or more nucleic acid fragments, the sequences can beassembled into a single contiguous nucleotide sequence, thus extendingthe original fragment in either the 5 or 3 prime direction. Once themost 5-prime EST is identified, its complete sequence can be determinedby Full Insert Sequencing as described in Example 1. Homologous genesbelonging to different species can be found by comparing the amino acidsequence of a known gene (from either a proprietary source or a publicdatabase) against an EST database using the tBLASTn algorithm. ThetBLASTn algorithm searches an amino acid query against a nucleotidedatabase that is translated in all 6 reading frames. This search allowsfor differences in nucleotide codon usage between different species, andfor codon degeneracy.

Example 3 Characterization of cDNA Clones Encoding Brittle-1 Homologs

[0084] The BLASTX search using the EST sequences from clonesbshl.pk0003.c5, rsl1n.pk013.j2, sfll.pk0015.h4, and ssm.pk0058.a1revealed similarity of the proteins encoded by the cDNAs to brittle-1homolog from Arabidopsis thaliana (NCBI GenBank Identifier (GI) No.4049342). The BLASTX search using the EST sequence from clonewdk1c.pk012.c23 revealed similarity of the protein encoded by the cDNAto brittle-1 protein from Zea mays (NCBI GI No. 231654). The BLASTXsearch using a different EST sequence from clone ssm.pk0058.a1 revealedsimilarity of the protein encoded by the cDNA to brittle-1 homolog fromSolanum tuberosum (NCBI GI No. 4138581). A BLASTP search using the aminoacid sequence encoded by the entire cDNA sequence from clonewre1n.pk0049.e1 revealed similarity of the encoded protein to brittle-1homolog from Solanum tuberosum (NCBI GI No. 4138581) (PCT PublicationNo. WO 99/49047).

[0085] The sequence of the entire cDNA insert in clones bsh1.pk0003.c5,sfl1.pk0015.h4, ssm.pk0058.a1, and wdk1c.pk012.c23 was determined. TheBLASTX search using the EST sequences from clones listed in Table 3revealed similarity of the polypeptides encoded by the cDNAs tobrittle-1 homologs from Arabidopsis thaliana (NCBI GenBank Identifier(GI) No. 7484793) and Solanum tuberosum (NCBI GI No. 4138581) andbrittle-1 protein from Zea mays (NCBI GI No. 231654). Shown in Table 3are the BLAST results for individual ESTs (“EST”), the sequences of theentire cDNA inserts comprising the indicated cDNA clones (“FIS”),sequences of contigs assembled from two or more ESTs (“Contig”),sequences of contigs assembled from an FIS and one or more ESTs(“Contig*”), or sequences encoding the entire protein derived from anFIS, a contig, or an FIS and PCR (“CGS”): TABLE 3 BLAST Results forSequences Encoding Polypeptides Homologous to Brittle-1 BLAST ResultsClone Status NCBI GI No. pLog Score bshl.pk0003.c5 FIS 7484793 46.70sfl1.pk0015.h4 (FIS) CGS 7484793 57.70 ssm.pk0058.a1 FIS 4138581 121.00wdk1c.pk012.c23 CGS 231654 152.00 (FIS)

[0086]FIG. 1 presents an alignment of the amino acid sequences set forthin SEQ ID NOs:10 and 18 and the Zea mays sequence (NCBI GI No. 231654;SEQ ID NO:21). The data in Table 4 represents a calculation of thepercent identity of the amino acid sequences set forth in SEQ ID NOs:10and 18 and the Zea mays sequence (NCBI GI No. 231654; SEQ ID NO:21).TABLE 4 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toBrittle-1 Protein Percent Identity to SEQ ID NO. NCBI GI No. 231654; SEQID NO: 21 10 27.1 18 57.3

[0087] Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores andprobabilities indicate that the nucleic acid fragments comprising theinstant cDNA clones encode a substantial portion of a brittle-1 homolog.These sequences represent the first barley, rice, soybean, and wheatsequences encoding brittle-1 homolog known to Applicant.

Example 4 Expression of Chimeric Genes in Monocot Cells

[0088] A chimeric gene comprising a cDNA encoding the instantpolypeptide in sense orientation with respect to the maize 27 kD zeinpromoter that is located 5′ to the cDNA fragment, and the 10 kD zein 3′end that is located 3′ to the cDNA fragment, can be constructed. ThecDNA fragment of this gene may be generated by polymerase chain reaction(PCR) of the cDNA clone using appropriate oligonucleotide primers.Cloning sites (NcoI or SmaI) can be incorporated into theoligonucleotides to provide proper orientation of the DNA fragment wheninserted into the digested vector pML103 as described below.Amplification is then performed in a standard PCR. The amplified DNA isthen digested with restriction enzymes NcoI and SmaI and fractionated onan agarose gel. The appropriate band can be isolated from the gel andcombined with a 4.9 kb NcoI-SmaI fragment of the plasmid pML103. PlasmidpML103 has been deposited under the terms of the Budapest Treaty at ATCC(American Type Culture Collection, 10801 University Blvd., Manassas, Va.20110-2209), and bears accession number ATCC 97366. The DNA segment frompML103 contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kDzein gene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize10 kD zein gene in the vector pGem9Zf(+) (Promega). Vector and insertDNA can be ligated at 15° C. overnight, essentially as described(Maniatis). The ligated DNA may then be used to transform E. coliXL1-Blue (Epicurian Coli XL-1 Blue™; Stratagene). Bacterialtransformants can be screened by restriction enzyme digestion of plasmidDNA and limited nucleotide sequence analysis using the dideoxy chaintermination method (Sequenase™ DNA Sequencing Kit; U.S. Biochemical).The resulting plasmid construct would comprise a chimeric gene encoding,in the 5′ to 3′ direction, the maize 27 kD zein promoter, a cDNAfragment encoding the instant polypeptide, and the 10 kD zein 3′ region.

[0089] The chimeric gene described above can then be introduced intocorn cells by the following procedure. Immature corn embryos can bedissected from developing caryopses derived from crosses of the inbredcorn lines H99 and LH132. The embryos are isolated 10 to 11 days afterpollination when they are 1.0 to 1.5 mm long. The embryos are thenplaced with the axis-side facing down and in contact withagarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking18:659-668). The embryos are kept in the dark at 27° C. Friableembryogenic callus consisting of undifferentiated masses of cells withsomatic proembryoids and embryoids borne on suspensor structuresproliferates from the scutellum of these immature embryos. Theembryogenic callus isolated from the primary explant can be cultured onN6 medium and sub-cultured on this medium every 2 to 3 weeks.

[0090] The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35 S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

[0091] The particle bombardment method (Klein et al. (1987) Nature327:70-73) may be used to transfer genes to the callus culture cells.According to this method, gold particles (1 μm in diameter) are coatedwith DNA using the following technique. Ten μg of plasmid DNAs are addedto 50 μL of a suspension of gold particles (60 mg per mL). Calciumchloride (50 μL of a 2.5 M solution) and spermidine free base (20 μL ofa 1.0 M solution) are added to the particles. The suspension is vortexedduring the addition of these solutions. After 10 minutes, the tubes arebriefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed.The particles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

[0092] For bombardment, the embryogenic tissue is placed on filter paperover agarose-solidified N6 medium. The tissue is arranged as a thin lawnand covered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

[0093] Seven days after bombardment the tissue can be transferred to N6medium that contains bialophos (5 mg per liter) and lacks casein orproline. The tissue continues to grow slowly on this medium. After anadditional 2 weeks the tissue can be transferred to fresh N6 mediumcontaining bialophos. After 6 weeks, areas of about 1 cm in diameter ofactively growing callus can be identified on some of the platescontaining the bialophos-supplemented medium. These calli may continueto grow when sub-cultured on the selective medium.

[0094] Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 5 Expression of Chimeric Genes in Dicot Cells

[0095] A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), Sma I, Kpn I and Xba I.The entire cassette is flanked by Hind III sites.

[0096] The cDNA fragment of this gene may be generated by polymerasechain reaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

[0097] Soybean embryos may then be transformed with the expressionvector comprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

[0098] Soybean embryogenic suspension cultures can be maintained in 35mL liquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 mL ofliquid medium.

[0099] Soybean embryogenic suspension cultures may then be transformedby the method of particle gun bombardment (Klein et al. (1987) Nature(London) 327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™PDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

[0100] A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al.(1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptide and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

[0101] To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μL spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

[0102] Approximately 300-400 mg of a two-week-old suspension culture isplaced in an empty 60×1 5 mm petri dish and the residual liquid removedfrom the tissue with a pipette. For each transformation experiment,approximately 5-10 plates of tissue are normally bombarded. Membranerupture pressure is set at 1100 psi and the chamber is evacuated to avacuum of 28 inches mercury. The tissue is placed approximately 3.5inches away from the retaining screen and bombarded three times.Following bombardment, the tissue can be divided in half and placed backinto liquid and cultured as described above.

[0103] Five to seven days post bombardment, the liquid media may beexchanged with fresh media, and eleven to twelve days post bombardmentwith fresh media containing 50 mg/mL hygromycin. This selective mediacan be refreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 6 Expression of Chimeric Genes in Microbial Cells

[0104] The cDNAs encoding the instant polypeptides can be inserted intothe T7 E. coli expression vector pBT430. This vector is a derivative ofpET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

[0105] Plasmid DNA containing a cDNA may be appropriately digested torelease a nucleic acid fragment encoding the protein. This fragment maythen be purified on a 1% low melting agarose gel. Buffer and agarosecontain 10 μg/ml ethidium bromide for visualization of the DNA fragment.The fragment can then be purified from the agarose gel by digestion withGELase™ (Epicentre Technologies, Madison, Wis.) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs (NEB), Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptide are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

[0106] For high level expression, a plasmid clone with the cDNA insertin the correct orientation relative to the T7 promoter can betransformed into E. coli strain BL21(DE3) (Studier et al. (1986) J. Mol.Biol 189:113-130). Cultures are grown in LB medium containing ampicillin(100 mg/L) at 25° C. At an optical density at 600 nm of approximately 1,IPTG (isopropylthio-β-galactoside, the inducer) can be added to a finalconcentration of 0.4 mM and incubation can be continued for 3 h at 25°.Cells are then harvested by centrifugation and re-suspended in 50 μL of50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride. A small amount of 1 mm glass beads can be addedand the mixture sonicated 3 times for about 5 seconds each time with amicroprobe sonicator. The mixture is centrifuged and the proteinconcentration of the supernatant determined. One μg of protein from thesoluble fraction of the culture can be separated by SDS-polyacrylamidegel electrophoresis. Gels can be observed for protein bands migrating atthe expected molecular weight.

1 21 1 539 DNA Hordeum vulgare 1 gcacgagcga gggctttggg tgggtaatacaatcaacatg atccgcatta ttcccactca 60 agcaattgag cttggaacat ttgagtatgtgaaaaggggc atgaggtcag cacaagagaa 120 atggaaagag gatggatgcc caaagatacagcttggtaat atgaacatcg agattccact 180 ccacttgtta tctccagttg ctattgctggtgcggccgct ggaatcgctg gcacattgat 240 gtgccatcct cttgaagtta ttaaggatcggctgaccgtg gatcgagtga cttatcctag 300 cattagcatt gccttcagca agatatatcgaactgaaggt atcagaggtc tctattctgg 360 cctctgccca acactaattg gcatgcttccttacagcaca tgctactact ttatgtacga 420 tacaatcaag acgtcgtact gccgcctacataagaagaaa tccttgagcc gtcctgagct 480 actaattata ggagctctga caagtctcacggcaagcacg atcagcttcc cgttggagg 539 2 112 PRT Hordeum vulgare 2 Ile AlaGly Ala Ala Ala Gly Ile Ala Gly Thr Leu Met Cys His Pro 1 5 10 15 LeuGlu Val Ile Lys Asp Arg Leu Thr Val Asp Arg Val Thr Tyr Pro 20 25 30 SerIle Ser Ile Ala Phe Ser Lys Ile Tyr Arg Thr Glu Gly Ile Arg 35 40 45 GlyLeu Tyr Ser Gly Leu Cys Pro Thr Leu Ile Gly Met Leu Pro Tyr 50 55 60 SerThr Cys Tyr Tyr Phe Met Tyr Asp Thr Ile Lys Thr Ser Tyr Cys 65 70 75 80Arg Leu His Lys Lys Lys Ser Leu Ser Arg Pro Glu Leu Leu Ile Ile 85 90 95Gly Ala Leu Thr Ser Leu Thr Ala Ser Thr Ile Ser Phe Pro Leu Glu 100 105110 3 1062 DNA Hordeum vulgare 3 gcacgagcga gggctttggg tgggtaatacaatcaacatg atccgcatta ttcccactca 60 agcaattgag cttggaacat ttgagtatgtgaaaaggggc atgaggtcag cacaagagaa 120 atggaaagag gatggatgcc caaagatacagcttggtaat atgaacatcg agattccact 180 ccacttgtta tctccagttg ctattgctggtgcggccgct ggaatcgctg gcacattgat 240 gtgccatcct cttgaagtta ttaaggatcggctgaccgtg gatcgagtga cttatcctag 300 cattagcatt gccttcagca agatatatcgaactgaaggt atcagaggtc tctattctgg 360 cctctgccca acactaattg gcatgcttccttacagcaca tgctactact ttatgtacga 420 tacaatcaag acgtcgtact gccgcctacataagaagaaa tccttgagcc gtcctgagct 480 actaattata ggagctctga caggtctcacggcaagcacg atcagcttcc cgttggaggt 540 ggcgaggaag cggctcatgg tgggcgccctgcaggggaag tgcccgccca acatggtggc 600 ggccctgtca gaagtgatcc gggaggagggcctcctgggg atctaccgtg ggtggggggc 660 gagctgcctc aaggtgatgc cgaattcgggcatcacctgg atgttctacg aggcgtggaa 720 ggatatcctc ctcgccgaga aggacaagcaccttgactag ccgacgatga tgatgaaaca 780 gcccttaggc ggaccactgg ccgaggatgatgagttactg aagaaacgaa cagtgctagg 840 cgttgctgta gtagaataat gtcgtgtggggggttctgat ccttttccag ttttcgaacc 900 ccacctctgc ctgtgccgaa ctgctagataggaggaactc gcggttgcaa ttgctgtcgg 960 gccgatgatg agtggaagca tttgtgtttgccgctccaaa aaaaaaaaaa aaaaaaaaaa 1020 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aa 1062 4 252 PRT Hordeum vulgare 4 His Glu Arg Gly Leu TrpVal Gly Asn Thr Ile Asn Met Ile Arg Ile 1 5 10 15 Ile Pro Thr Gln AlaIle Glu Leu Gly Thr Phe Glu Tyr Val Lys Arg 20 25 30 Gly Met Arg Ser AlaGln Glu Lys Trp Lys Glu Asp Gly Cys Pro Lys 35 40 45 Ile Gln Leu Gly AsnMet Asn Ile Glu Ile Pro Leu His Leu Leu Ser 50 55 60 Pro Val Ala Ile AlaGly Ala Ala Ala Gly Ile Ala Gly Thr Leu Met 65 70 75 80 Cys His Pro LeuGlu Val Ile Lys Asp Arg Leu Thr Val Asp Arg Val 85 90 95 Thr Tyr Pro SerIle Ser Ile Ala Phe Ser Lys Ile Tyr Arg Thr Glu 100 105 110 Gly Ile ArgGly Leu Tyr Ser Gly Leu Cys Pro Thr Leu Ile Gly Met 115 120 125 Leu ProTyr Ser Thr Cys Tyr Tyr Phe Met Tyr Asp Thr Ile Lys Thr 130 135 140 SerTyr Cys Arg Leu His Lys Lys Lys Ser Leu Ser Arg Pro Glu Leu 145 150 155160 Leu Ile Ile Gly Ala Leu Thr Gly Leu Thr Ala Ser Thr Ile Ser Phe 165170 175 Pro Leu Glu Val Ala Arg Lys Arg Leu Met Val Gly Ala Leu Gln Gly180 185 190 Lys Cys Pro Pro Asn Met Val Ala Ala Leu Ser Glu Val Ile ArgGlu 195 200 205 Glu Gly Leu Leu Gly Ile Tyr Arg Gly Trp Gly Ala Ser CysLeu Lys 210 215 220 Val Met Pro Asn Ser Gly Ile Thr Trp Met Phe Tyr GluAla Trp Lys 225 230 235 240 Asp Ile Leu Leu Ala Glu Lys Asp Lys His LeuAsp 245 250 5 436 DNA Oryza sativa unsure (126) unsure (184) unsure(302) unsure (307) unsure (325) unsure (352) unsure (372) unsure(379)..(380) unsure (392) unsure (399) unsure (403) unsure (407) unsure(418)..(419) unsure (422) unsure (436) 5 gcacggcaat aagaagtcttgatcgtctca taaaaagaag tccttgagcc gtcctgagct 60 actcattatt ggagctctttcaggtctaac tgcaagtaca ataagcttcc ctctggaagt 120 ggcgangaag cggcttatggttggaaccct gcaagggaaa tgcccgcccc acatgatcgc 180 ggtncttagc tgaggtgttccaagaggagg gcatcaaggg actttaccgc ggatgggccg 240 caagctccct gaaggtgatgccgacctccg gcattacctg gatgttctat ggagggatgg 300 gnagggncat tccttttgggctccnagagc ctgcacaccc taagccacct angcaaggtc 360 ctaagccaat gnccctggnnaatttgtttt cnaacaatng gcnaggngaa tttggctnnc 420 angaaataaa attttn 436 646 PRT Oryza sativa UNSURE (35) 6 Ser Ser His Lys Lys Lys Ser Leu SerArg Pro Glu Leu Leu Ile Ile 1 5 10 15 Gly Ala Leu Ser Gly Leu Thr AlaSer Thr Ile Ser Phe Pro Leu Glu 20 25 30 Val Ala Xaa Lys Arg Leu Met ValGly Thr Leu Gln Gly Lys 35 40 45 7 1517 DNA Glycine max 7 gcacgaggaaaggaacccta ataaacatat tgtgatcttc gaagcaacaa taataaagag 60 cagagtagagaagtgaaaaa caattttaca gaacccactc ggccccaaat aaaaaacaaa 120 atgtcgtcttccaactccaa aaccaaaacc ccttcttcac tctcactctg caactctaag 180 cctcagcctcaggaaggtaa catggcattg gaatcccaac cgcagaagaa caagtatgga 240 cacggggtgtttggagacgt ctacagcatc atcaaagaga tggagattga tcatcataac 300 aactctacctttgattttca atttccccca attacaaatt ttcttggctc tagagaggtt 360 cgggagtttattagcggggc cctttcaggg gcaatgacaa aggtatactt gctcctcttg 420 agaccatcaggacaagaatg tagttgtgtt ggtcagaaaa tattgctgta gtttcataga 480 ggttatagagcagcagggat ggcaaggact gtgggctgga aacatgatca atatgcttcg 540 tatagttccaacacaggcca ttgagctagg cacatttgag tgtgtcaaac gggctatgac 600 atccctgcatgagaaatggg aaagcaatga ataccccaag ttgcagatag gtcccatcaa 660 tttcaacttatctttatctt ggatttcacc agttgccatc gccggtgcag ctgctggaat 720 tgctagcactcttgtatgcc atccccttga agttttgaag gaccggttaa ctgtaagtcc 780 tgaaacttaccctagtttag gcattgcgat tagaaatatt tataaagacg gaggtgttgg 840 cgctttttatgctggtatct caccaactct ggttggcatg cttccataca gtacatgttt 900 ttatttcatgtatgatacaa taaaggaatc ttactgccgg accaaaagta agaaatctct 960 aagccgtccagagatgcttt tgattggagc tcttgcaggt tttactgcca gtacaattag 1020 cttccccttggaggtagcaa ggaagcgcct gatggtgggt gctttgcaag gtaagtgccc 1080 gccaaacatggcagcggcac tttcagaagt tattagagaa gaaggtctga agggtctcta 1140 cagaggatggggtgcaagct gtttgaaggt catgccatcc tctggtatca cctggatgtt 1200 ttatgaagcttggaaagaca tattgcttgt ccagaatggt aatccccttt aggctaataa 1260 gaaggttatgcggataactt gtactctata agaaggaatg gtaaaacaca atgccgggta 1320 aggaaaaataattattcgga aattaaagtt gttttgccat ggcagccagc tggctagctc 1380 gttggaatagtcacatttac taacgagtta ggtacctagc ttgtaagttt ccagattttg 1440 atattgatttgggaagattc tcatctaagt taaaggttaa ggcggggcaa aataaattct 1500 cgggccgggccataacc 1517 8 180 PRT Glycine max 8 Ile Ala Gly Ala Ala Ala Gly Ile AlaSer Thr Leu Val Cys His Pro 1 5 10 15 Leu Glu Val Leu Lys Asp Arg LeuThr Val Ser Pro Glu Thr Tyr Pro 20 25 30 Ser Leu Gly Ile Ala Ile Arg AsnIle Tyr Lys Asp Gly Gly Val Gly 35 40 45 Ala Phe Tyr Ala Gly Ile Ser ProThr Leu Val Gly Met Leu Pro Tyr 50 55 60 Ser Thr Cys Phe Tyr Phe Met TyrAsp Thr Ile Lys Glu Ser Tyr Cys 65 70 75 80 Arg Thr Lys Ser Lys Lys SerLeu Ser Arg Pro Glu Met Leu Leu Ile 85 90 95 Gly Ala Leu Ala Gly Phe ThrAla Ser Thr Ile Ser Phe Pro Leu Glu 100 105 110 Val Ala Arg Lys Arg LeuMet Val Gly Ala Leu Gln Gly Lys Cys Pro 115 120 125 Pro Asn Met ala AlaAla Leu Ser Glu Val Ile Arg Glu Glu Gly Leu 130 135 140 Lys Gly Leu TyrArg Gly Trp Gly Ala Ser Cys Leu Lys Val Met Pro 145 150 155 160 Ser SerGly Ile Thr Trp Met Phe Tyr Glu Ala Trp Lys Asp Ile Leu 165 170 175 LeuVal Gln Asn 180 9 1506 DNA Glycine max 9 gcacgaggaa aggaaccctaataaacatat tgtgatcttc gaagcaacaa taataaagag 60 cagagtagag aagtgaaaaacaattttaca gaacccactc ggccccaaat aaaaaacaaa 120 atgtcgtctt ccaactccaaaaccaaaacc ccttcttcac tctcactctg caactctaag 180 cctcagcctc aggaaggtaacatggcattg gaatcccaac cgcagaagaa caagtatgga 240 cacggggtgt ttggagacgtctacagcatc atcaaagaga tggagattga tcatcataac 300 aactctacct ttgattttcaatttccccca attacaaatt ttcttggctc tagagaggtt 360 cgggagttta ttagcggggccctttcaggg gcaatgacaa aggctatact tgctcctctt 420 gagaccatca ggacaagaatggtagttggt gttgggtcaa aaaatattgc tggtagtttc 480 atagaggtta tagagcagcagggatggcaa ggactgtggg ctggaaacat gatcaatatg 540 cttcgtatag ttccaacacaggccattgag ctaggcacat ttgagtgtgt caaacgggct 600 atgacatccc tgcatgagaaatgggaaagc aatgaatacc ccaagttgca gataggtccc 660 atcaatttca acttatctttatcttggatt tcaccagttg ccatcgccgg tgcagctgct 720 ggaattgcta gcactcttgtatgccatccc cttgaagttt tgaaggaccg gttaactgta 780 agtcctgaaa cttaccctagtttaggcatt gcgattagaa atatttataa agacggaggt 840 gttggcgctt tttatgctggtatctcacca actctggttg gcatgcttcc atacagtaca 900 tgtttttatt tcatgtatgatacaataaag gaatcttact gccggaccaa aagtaagaaa 960 tctctaagcc gtccagagatgcttttgatt ggagctcttg caggttttac tgccagtaca 1020 attagcttcc ccttggaggtagcaaggaag cgcctgatgg tgggtgcttt gcaaggtaag 1080 tgcccgccaa acatggcagcggcactttca gaagttatta gagaagaagg tctgaagggt 1140 ctctacagag gatggggtgcaagctgtttg aaggtcatgc catcctctgg tatcacctgg 1200 atgttttatg aagcttggaaagacatattg cttgtccaga atggtaatcc cctttaggct 1260 aataagaagg ttatgcggataacttgtact ctataagaag gaatggtaaa acacaatgcc 1320 gggtaaggaa aaataattattcggaaatta aagttgtttt gccatggcag ccagctggct 1380 agctcgttgg aatagtcacatttactaacg agttaggtac ctagcttgta agtttccaga 1440 ttttgatatt gatttgggaagattctcatc gaagaaaaag gttaaagaaa aaaaaaaaaa 1500 aaaaaa 1506 10 410 PRTGlycine max 10 Thr Tyr Cys Asp Leu Arg Ser Asn Asn Asn Lys Glu Gln SerArg Glu 1 5 10 15 Val Lys Asn Asn Phe Thr Glu Pro Thr Arg Pro Gln IleLys Asn Lys 20 25 30 Met Ser Ser Ser Asn Ser Lys Thr Lys Thr Pro Ser SerLeu Ser Leu 35 40 45 Cys Asn Ser Lys Pro Gln Pro Gln Glu Gly Asn Met AlaLeu Glu Ser 50 55 60 Gln Pro Gln Lys Asn Lys Tyr Gly His Gly Val Phe GlyAsp Val Tyr 65 70 75 80 Ser Ile Ile Lys Glu Met Glu Ile Asp His His AsnAsn Ser Thr Phe 85 90 95 Asp Phe Gln Phe Pro Pro Ile Thr Asn Phe Leu GlySer Arg Glu Val 100 105 110 Arg Glu Phe Ile Ser Gly Ala Leu Ser Gly AlaMet Thr Lys Ala Ile 115 120 125 Leu Ala Pro Leu Glu Thr Ile Arg Thr ArgMet Val Val Gly Val Gly 130 135 140 Ser Lys Asn Ile Ala Gly Ser Phe IleGlu Val Ile Glu Gln Gln Gly 145 150 155 160 Trp Gln Gly Leu Trp Ala GlyAsn Met Ile Asn Met Leu Arg Ile Val 165 170 175 Pro Thr Gln Ala Ile GluLeu Gly Thr Phe Glu Cys Val Lys Arg Ala 180 185 190 Met Thr Ser Leu HisGlu Lys Trp Glu Ser Asn Glu Tyr Pro Lys Leu 195 200 205 Gln Ile Gly ProIle Asn Phe Asn Leu Ser Leu Ser Trp Ile Ser Pro 210 215 220 Val Ala IleAla Gly Ala Ala Ala Gly Ile Ala Ser Thr Leu Val Cys 225 230 235 240 HisPro Leu Glu Val Leu Lys Asp Arg Leu Thr Val Ser Pro Glu Thr 245 250 255Tyr Pro Ser Leu Gly Ile Ala Ile Arg Asn Ile Tyr Lys Asp Gly Gly 260 265270 Val Gly Ala Phe Tyr Ala Gly Ile Ser Pro Thr Leu Val Gly Met Leu 275280 285 Pro Tyr Ser Thr Cys Phe Tyr Phe Met Tyr Asp Thr Ile Lys Glu Ser290 295 300 Tyr Cys Arg Thr Lys Ser Lys Lys Ser Leu Ser Arg Pro Glu MetLeu 305 310 315 320 Leu Ile Gly Ala Leu Ala Gly Phe Thr Ala Ser Thr IleSer Phe Pro 325 330 335 Leu Glu Val Ala Arg Lys Arg Leu Met Val Gly AlaLeu Gln Gly Lys 340 345 350 Cys Pro Pro Asn Met Ala Ala Ala Leu Ser GluVal Ile Arg Glu Glu 355 360 365 Gly Leu Lys Gly Leu Tyr Arg Gly Trp GlyAla Ser Cys Leu Lys Val 370 375 380 Met Pro Ser Ser Gly Ile Thr Trp MetPhe Tyr Glu Ala Trp Lys Asp 385 390 395 400 Ile Leu Leu Val Gln Asn GlyAsn Pro Leu 405 410 11 504 DNA Glycine max 11 gtgtgcacca ttcccacttgaggtggttgt taagcatatg caagctgggg ctttaaatga 60 aagacaatat gggaacatgcttcatgcact tgtgagtata cttaaaaagg aaggagttgg 120 tggcttgtat agaggtttgggaccaagttg cttaaaattg gttcctgctg ctgggatttc 180 tttcatgtgc tacgaagcttgcaagaggat acttgttgaa aatgaacaag attaattaca 240 agtggatcac tgcatattctttccatggga tatattggca ttgttttgtg tttttgaaga 300 gggaaataat ttgtcgagctaatttttggt tttgcagatt ttgcttttcc ttgcatattt 360 gaccatttca actagggtgtttcttttaag ttgcattggc tttaaggaaa aaagttgtat 420 tgattacaga ctctaatttattttacaatc aattgtgttt ctttcccaga aaaaaaaaaa 480 aaaaaaaaaa aaaaaaaaaaaaaa 504 12 76 PRT Glycine max 12 Ala Pro Phe Pro Leu Glu Val Val ValLys His Met Gln Ala Gly Ala 1 5 10 15 Leu Asn Glu Arg Gln Tyr Gly AsnMet Leu His Ala Leu Val Ser Ile 20 25 30 Leu Lys Lys Glu Gly Val Gly GlyLeu Tyr Arg Gly Leu Gly Pro Ser 35 40 45 Cys Leu Lys Leu Val Pro Ala AlaGly Ile Ser Phe Met Cys Tyr Glu 50 55 60 Ala Cys Lys Arg Ile Leu Val GluAsn Glu Gln Asp 65 70 75 13 1089 DNA Glycine max 13 gcacgaggtgcagtgtcaag gacagccgtg gcaccgttgg aaaccataag gactcatttg 60 atggtggggagctgtgggca tagtacaatt caagtgtttc aatctattat ggagaccgat 120 ggatggaagggcttgttcag aggcaatttt gtaaacatca tccgagttgc gccaagcaag 180 gccattgagttatttgcata tgacactgtc aagaagcaat tatctccgaa acctggagag 240 cagcctataatcccaattcc cccctcatca attgcgggtg ctgttgctgg tgttagctct 300 accctatgtacataccctct tgaactactc aaaactcgcc tcactgttca gagaggggtg 360 tacaagaacttactcgacgc atttgtgagg atcgttcaag aggaaggtcc tgcagaattg 420 tataggggcctcgcccctag tctaattggt gtaatccctt atgctgcaac aaactacttt 480 gcttatgacacacttagaaa agcttacaag aaagctttca aaaaggagga gattgggaat 540 gtgatgactcttctaattgg atcagctgct ggtgcaattt cgagtagtgc aacatttcca 600 cttgaggtggctcgtaagca tatgcaagct ggggctctaa atggaagaca atatgggaac 660 atgcttcatgcacttgtgag tatacttgaa aaggaaggag ttggtggctt gtatagaggt 720 ttgggaccaagttgcttaaa attggttcct gctgctggga tttctttcat gtgctacgaa 780 gcttgcaagaggatacttgt tgaaaatgaa caagattaat tacaagtgga tcactgcata 840 ttctttccatgggatatatt ggcattgttt tgtgtttttg aagagggaaa taatttgtcg 900 agctaatttttggttttgca gattttgctt ttccttgcat atttgaccat ttcaactagg 960 gtgtttcttttaagttgcat tggctttaag gaaaaaagtt gtattgatta cagactctaa 1020 tttattttacaatcaattgt gtttcataaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1080 aaaaaaaaa1089 14 272 PRT Glycine max 14 Ala Arg Gly Ala Val Ser Arg Thr Ala ValAla Pro Leu Glu Thr Ile 1 5 10 15 Arg Thr His Leu Met Val Gly Ser CysGly His Ser Thr Ile Gln Val 20 25 30 Phe Gln Ser Ile Met Glu Thr Asp GlyTrp Lys Gly Leu Phe Arg Gly 35 40 45 Asn Phe Val Asn Ile Ile Arg Val AlaPro Ser Lys Ala Ile Glu Leu 50 55 60 Phe Ala Tyr Asp Thr Val Lys Lys GlnLeu Ser Pro Lys Pro Gly Glu 65 70 75 80 Gln Pro Ile Ile Pro Ile Pro ProSer Ser Ile Ala Gly Ala Val Ala 85 90 95 Gly Val Ser Ser Thr Leu Cys ThrTyr Pro Leu Glu Leu Leu Lys Thr 100 105 110 Arg Leu Thr Val Gln Arg GlyVal Tyr Lys Asn Leu Leu Asp Ala Phe 115 120 125 Val Arg Ile Val Gln GluGlu Gly Pro Ala Glu Leu Tyr Arg Gly Leu 130 135 140 Ala Pro Ser Leu IleGly Val Ile Pro Tyr Ala Ala Thr Asn Tyr Phe 145 150 155 160 Ala Tyr AspThr Leu Arg Lys Ala Tyr Lys Lys Ala Phe Lys Lys Glu 165 170 175 Glu IleGly Asn Val Met Thr Leu Leu Ile Gly Ser Ala Ala Gly Ala 180 185 190 IleSer Ser Ser Ala Thr Phe Pro Leu Glu Val Ala Arg Lys His Met 195 200 205Gln Ala Gly Ala Leu Asn Gly Arg Gln Tyr Gly Asn Met Leu His Ala 210 215220 Leu Val Ser Ile Leu Glu Lys Glu Gly Val Gly Gly Leu Tyr Arg Gly 225230 235 240 Leu Gly Pro Ser Cys Leu Lys Leu Val Pro Ala Ala Gly Ile SerPhe 245 250 255 Met Cys Tyr Glu Ala Cys Lys Arg Ile Leu Val Glu Asn GluGln Asp 260 265 270 15 449 DNA Triticum aestivum unsure (6) unsure (268)unsure (383) unsure (449) 15 ggccantgag ggagtgaagg actgaagaac tcctaggcagggcacgtatc agttctgtct 60 tgcttcctcg aagatggcgg cggcaatggc cgcgacgacaatggtgacca agaacaaccg 120 cgcctcgctc gtcatggaca agaagaactg gttattgcggccggtccctg aggtcgcctt 180 cccttggagc tcgcagcccg agtccaggag cttggacttcccacgcaggg ctctgttcgc 240 cagcgtggga ctcagcctgt cccacggngc cccgccggtagcgcgcgagc atgacgggaa 300 ggctcggccc gccgacgacg tctcacacca agctcgcatccgcgggcgag gcgggcgtcc 360 agaaggccca gaaggcgaaa aanggcaaaa agcagcagctgagtctgaag gaaggtgagg 420 ggtcaagatc ggcaacccgc acctgcggn 449 16 109 PRTTriticum aestivum UNSURE (104) 16 Met Ala Ala Ala Met Ala Ala Thr ThrMet Val Thr Lys Asn Asn Arg 1 5 10 15 Ala Ser Leu Val Met Asp Lys LysAsn Trp Leu Leu Arg Pro Val Pro 20 25 30 Glu Val Ala Phe Pro Trp Ser SerGln Pro Glu Ser Arg Ser Leu Asp 35 40 45 Phe Pro Arg Arg Ala Leu Phe AlaSer Val Gly Leu Ser Leu Ser His 50 55 60 Gly Ala Pro Pro Val Ala Arg GluHis Asp Gly Lys Ala Arg Pro Ala 65 70 75 80 Asp Asp Val Ser His Gln AlaArg Ile Arg Gly Arg Gly Gly Arg Pro 85 90 95 Glu Gly Pro Glu Gly Glu LysXaa Gln Lys Ala Ala Ala 100 105 17 1625 DNA Triticum aestivum 17ggccagtgag ggagtgaagg actgaagaac tcctaggcag ggcacgtatc agttctgtct 60tgcttcctcg agatggcggc ggcaatggcc gcgacgacaa tggtgaccaa gaacaaccgc 120gcctcgctcg tcatggacaa gaagaactgg ttattgcggc cggtccctga ggtcgccttc 180ccttggagct cgcagcccga gtccaggagc ttggacttcc cacgcagggc tctgttcgcc 240agcgtgggac tcagcctgtc ccacggcgcc ccgccggtag cgcgcgagca tgacgggaag 300gctcggcccg ccgacgacgt cgcacaccag ctcgcagccg cgggcgaggc gggcgtccag 360aaggcccaga aggcgaaaaa ggccaaaaag cagcagctga gtctgaggaa ggtgagggtc 420aagatcggca acccgcacct gcggcggctg gtcagcggcg ccatcgccgg cgccgtgtcg 480aggactttcg tggcgccact ggagacgatc aggacgcacc tgatggtggg gagctccggc 540gccgactcca tggccggggt tttccggtgg atcatgcgga cggaggggtg gcccggcctc 600ttccgcggca acgccgtcaa cgtcctccgc gtcgcgccaa gcaaggccat cgagcacttc 660acttacgaca cggcgaagaa gtacctgacc ccggaggccg gcgagccagc caaggtcccc 720atccccacgc cgctcgtcgc cggagcgctc gccggagtgg cgtcaaccct gtgcacctat 780cccatggagc tcgtcaagac ccgtctcacc atcgagaagg acgtgtacga caacctcctc 840cacgcgttcg tcaagatcgt gcgcgacgaa ggcccggggg agctgtaccg cgggctggcg 900ccgagcctga tcggcgtggt gccgtacgcg gcggccaact tctacgccta cgagacgctg 960cgcggcgtgt accgccgcgc gtcggggaaa gaggaggtgg gcaacgtccc gacgctgctg 1020atcgggtccg cggcgggcgc catagccagc acggccacgt tcccgctgga ggtggcgcgg 1080aagcagatgc aggtgggcgc cgtgggcggg aggcaggtgt acaagaacgt gctgcacgcc 1140atgtactgca tcctcgagaa ggagggcacc gccgggctct accgcgggct cggccccagc 1200tgcatcaagc tcatgcccgc cgccggcatc tccttcatgt gctacgaggc ctgcaagaag 1260atacttgtcg acgagaaaga agacggcggc gccgccgagc cccaggagga gacggagacc 1320ggacaggcag gaggacaggc ggcgcccaag agctcgaacg gtgatcggcc atgaactaga 1380tgaagcatta tggtgaccgt caaaatcaga agaaaatgcg tgatttgaaa tttttgaagt 1440gtagagccta ttgcgattga atcctaagct ggaagtggcg ccttagaagt tgaatttcgt 1500tttgttcagg gaacatgctc cgtttcagta atgccgtcga atgatttatg gcacctttct 1560gtaatcaatt caataaggaa gaagtccact tttggacctt caaaaaaaaa aaaaaaaaaa 1620aaaaa 1625 18 433 PRT Triticum aestivum 18 Met Ala Ala Ala Met Ala AlaThr Thr Met Val Thr Lys Asn Asn Arg 1 5 10 15 Ala Ser Leu Val Met AspLys Lys Asn Trp Leu Leu Arg Pro Val Pro 20 25 30 Glu Val Ala Phe Pro TrpSer Ser Gln Pro Glu Ser Arg Ser Leu Asp 35 40 45 Phe Pro Arg Arg Ala LeuPhe Ala Ser Val Gly Leu Ser Leu Ser His 50 55 60 Gly Ala Pro Pro Val AlaArg Glu His Asp Gly Lys Ala Arg Pro Ala 65 70 75 80 Asp Asp Val Ala HisGln Leu Ala Ala Ala Gly Glu Ala Gly Val Gln 85 90 95 Lys Ala Gln Lys AlaLys Lys Ala Lys Lys Gln Gln Leu Ser Leu Arg 100 105 110 Lys Val Arg ValLys Ile Gly Asn Pro His Leu Arg Arg Leu Val Ser 115 120 125 Gly Ala IleAla Gly Ala Val Ser Arg Thr Phe Val Ala Pro Leu Glu 130 135 140 Thr IleArg Thr His Leu Met Val Gly Ser Ser Gly Ala Asp Ser Met 145 150 155 160Ala Gly Val Phe Arg Trp Ile Met Arg Thr Glu Gly Trp Pro Gly Leu 165 170175 Phe Arg Gly Asn Ala Val Asn Val Leu Arg Val Ala Pro Ser Lys Ala 180185 190 Ile Glu His Phe Thr Tyr Asp Thr Ala Lys Lys Tyr Leu Thr Pro Glu195 200 205 Ala Gly Glu Pro Ala Lys Val Pro Ile Pro Thr Pro Leu Val AlaGly 210 215 220 Ala Leu Ala Gly Val Ala Ser Thr Leu Cys Thr Tyr Pro MetGlu Leu 225 230 235 240 Val Lys Thr Arg Leu Thr Ile Glu Lys Asp Val TyrAsp Asn Leu Leu 245 250 255 His Ala Phe Val Lys Ile Val Arg Asp Glu GlyPro Gly Glu Leu Tyr 260 265 270 Arg Gly Leu Ala Pro Ser Leu Ile Gly ValVal Pro Tyr Ala Ala Ala 275 280 285 Asn Phe Tyr Ala Tyr Glu Thr Leu ArgGly Val Tyr Arg Arg Ala Ser 290 295 300 Gly Lys Glu Glu Val Gly Asn ValPro Thr Leu Leu Ile Gly Ser Ala 305 310 315 320 Ala Gly Ala Ile Ala SerThr Ala Thr Phe Pro Leu Glu Val Ala Arg 325 330 335 Lys Gln Met Gln ValGly Ala Val Gly Gly Arg Gln Val Tyr Lys Asn 340 345 350 Val Leu His AlaMet Tyr Cys Ile Leu Glu Lys Glu Gly Thr Ala Gly 355 360 365 Leu Tyr ArgGly Leu Gly Pro Ser Cys Ile Lys Leu Met Pro Ala Ala 370 375 380 Gly IleSer Phe Met Cys Tyr Glu Ala Cys Lys Lys Ile Leu Val Asp 385 390 395 400Glu Lys Glu Asp Gly Gly Ala Ala Glu Pro Gln Glu Glu Thr Glu Thr 405 410415 Gly Gln Ala Gly Gly Gln Ala Ala Pro Lys Ser Ser Asn Gly Asp Arg 420425 430 Pro 19 1267 DNA Triticum aestivum 19 caagattaag gttgggaattcacacctcaa gaggctcatc agtgggggga ttgcaggagc 60 agtgtcaagg acagttgtggcgcctttgga gacgattagg acacatttga tggtcggcag 120 caatgggaat tcatctacggaggtgtttga ctccatcatg aagaatgaag gatggactgg 180 gttgttccgc ggcaatttggttaatgtcat tcgagtcgcc ccgagcaaag caatcgagct 240 ttttgccttt gatacagctaagaagttcct aacccccaaa tctggggaag aacagaagat 300 cccaatccct ccttcactagtggcaggagc ttttgctggt gtcagctcaa ctctgtgtac 360 ataccctctg gaactaattaagactcgatt aaccatacag agaggtgtgt atgataactt 420 cctccatgca tttgtgaaaattgtccgtga agaaggccct gctgagctgt atagaggctt 480 aaccccaagt ctaatcggagtggtgccata tgcagcaacc aactacttcg cgtatgacac 540 ccttaagaag gtgtacaagaaaatgttcaa gacaaatgaa atcggcaacg ttccaaccct 600 gctcattggg tctgctgcaggagccatctc aagcactgcc acatttcctc tcgaggttgc 660 tcgcaagcac atgcaagtcggagctgttgg cggccggaag gtatacaaga acatgcttca 720 cgctctcctg accattctcgaggacgaagg ggttgggggc ctctacagag gactggggcc 780 tagttgcatg aagctggtgcctgctgctgg gatttcgttt atgtgctacg aagcttgcaa 840 gaagatactg attgaggaagagaacgaatg aagcgttctt caacagcggc gtcataaagg 900 ggtagtggct taaattttgtttgctgatcc tatgatggat ctgaatctga tcctggggcc 960 ttcctcccaa gataccagagctcggtttcg cgacggacag cggggaaact tttggcctcc 1020 tttgaatgaa gttacctgactaagctcaat aactgttgct acaagatttc aaactctttc 1080 tttagtctca gcttgccctgacaaaaagtt acatatgttt ccagtttgct ttgggatact 1140 atatgcatga atgaagcgtgtgttttttag gaagtctttg ttagggggta tatgaaacca 1200 gtgaaattaa ctccggagacatcaaatttt acatgattga catcaaaaaa aaaaaaaaaa 1260 aaaaaaa 1267 20 289 PRTTriticum aestivum 20 Lys Ile Lys Val Gly Asn Ser His Leu Lys Arg Leu IleSer Gly Gly 1 5 10 15 Ile Ala Gly Ala Val Ser Arg Thr Val Val Ala ProLeu Glu Thr Ile 20 25 30 Arg Thr His Leu Met Val Gly Ser Asn Gly Asn SerSer Thr Glu Val 35 40 45 Phe Asp Ser Ile Met Lys Asn Glu Gly Trp Thr GlyLeu Phe Arg Gly 50 55 60 Asn Leu Val Asn Val Ile Arg Val Ala Pro Ser LysAla Ile Glu Leu 65 70 75 80 Phe Ala Phe Asp Thr Ala Lys Lys Phe Leu ThrPro Lys Ser Gly Glu 85 90 95 Glu Gln Lys Ile Pro Ile Pro Pro Ser Leu ValAla Gly Ala Phe Ala 100 105 110 Gly Val Ser Ser Thr Leu Cys Thr Tyr ProLeu Glu Leu Ile Lys Thr 115 120 125 Arg Leu Thr Ile Gln Arg Gly Val TyrAsp Asn Phe Leu His Ala Phe 130 135 140 Val Lys Ile Val Arg Glu Glu GlyPro Ala Glu Leu Tyr Arg Gly Leu 145 150 155 160 Thr Pro Ser Leu Ile GlyVal Val Pro Tyr Ala Ala Thr Asn Tyr Phe 165 170 175 Ala Tyr Asp Thr LeuLys Lys Val Tyr Lys Lys Met Phe Lys Thr Asn 180 185 190 Glu Ile Gly AsnVal Pro Thr Leu Leu Ile Gly Ser Ala Ala Gly Ala 195 200 205 Ile Ser SerThr Ala Thr Phe Pro Leu Glu Val Ala Arg Lys His Met 210 215 220 Gln ValGly Ala Val Gly Gly Arg Lys Val Tyr Lys Asn Met Leu His 225 230 235 240Ala Leu Leu Thr Ile Leu Glu Asp Glu Gly Val Gly Gly Leu Tyr Arg 245 250255 Gly Leu Gly Pro Ser Cys Met Lys Leu Val Pro Ala Ala Gly Ile Ser 260265 270 Phe Met Cys Tyr Glu Ala Cys Lys Lys Ile Leu Ile Glu Glu Glu Asn275 280 285 Glu 21 436 PRT Zea mays 21 Met Ala Ala Thr Met Ala Val ThrThr Met Val Thr Arg Ser Lys Glu 1 5 10 15 Ser Trp Ser Ser Leu Gln ValPro Ala Val Ala Phe Pro Trp Lys Pro 20 25 30 Arg Gly Gly Lys Thr Gly GlyLeu Glu Phe Pro Arg Arg Ala Met Phe 35 40 45 Ala Ser Val Gly Leu Asn ValCys Pro Gly Val Pro Ala Gly Arg Asp 50 55 60 Pro Arg Glu Pro Asp Pro LysVal Val Arg Ala Ala Asp Asn Cys Asp 65 70 75 80 Ile Ala Ala Ser Leu AlaPro Pro Phe Pro Gly Ser Arg Pro Pro Gly 85 90 95 Arg Arg Gly Arg Gly SerGlu Glu Glu Glu Ala Glu Gly Arg Arg His 100 105 110 Glu Glu Ala Ala AlaAla Gly Arg Ser Glu Pro Glu Glu Gly Gln Gly 115 120 125 Gln Asp Arg GlnPro Ala Pro Ala Arg Leu Val Ser Gly Ala Ile Ala 130 135 140 Gly Ala ValSer Arg Thr Phe Val Ala Pro Leu Glu Thr Ile Arg Thr 145 150 155 160 HisLeu Met Val Gly Ser Ile Gly Val Asp Ser Met Ala Gly Val Phe 165 170 175Gln Trp Ile Met Gln Asn Glu Gly Trp Thr Gly Leu Phe Arg Gly Asn 180 185190 Ala Val Asn Val Leu Arg Val Ala Pro Ser Lys Ala Ile Glu His Phe 195200 205 Thr Tyr Asp Thr Ala Lys Lys Phe Leu Thr Pro Lys Gly Asp Glu Pro210 215 220 Pro Lys Ile Pro Ile Pro Thr Pro Leu Val Ala Gly Ala Leu AlaGly 225 230 235 240 Phe Ala Ser Thr Leu Cys Thr Tyr Pro Met Glu Leu IleLys Thr Arg 245 250 255 Val Thr Ile Glu Lys Asp Val Tyr Asp Asn Val AlaHis Ala Phe Val 260 265 270 Lys Ile Leu Arg Asp Glu Gly Pro Ser Glu LeuTyr Arg Gly Leu Thr 275 280 285 Pro Ser Leu Ile Gly Val Val Pro Tyr AlaAla Cys Asn Phe Tyr Ala 290 295 300 Tyr Glu Thr Leu Lys Arg Leu Tyr ArgArg Ala Thr Gly Arg Arg Pro 305 310 315 320 Gly Ala Asp Val Gly Pro ValAla Thr Leu Leu Ile Gly Ser Ala Ala 325 330 335 Gly Ala Ile Ala Ser SerAla Thr Phe Pro Leu Glu Val Ala Arg Lys 340 345 350 Gln Met Gln Val GlyAla Val Gly Gly Arg Gln Val Tyr Gln Asn Val 355 360 365 Leu His Ala IleTyr Cys Ile Leu Lys Lys Glu Gly Ala Gly Gly Leu 370 375 380 Tyr Arg GlyLeu Gly Pro Ser Cys Ile Lys Leu Met Pro Ala Ala Gly 385 390 395 400 IleAla Phe Met Cys Tyr Glu Ala Cys Lys Lys Ile Leu Val Asp Lys 405 410 415Glu Asp Glu Glu Glu Glu Asp Glu Ala Gly Gly Gly Glu Asp Asp Lys 420 425430 Lys Lys Val Glu 435

What is claimed is:
 1. An isolated polynucleotide comprising: (a) afirst nucleotide sequence encoding a first polypeptide comprising atleast 200 amino acids, wherein the amino acid sequence of the firstpolypeptide and the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:10have at least 70% identity based on the Clustal alignment method, (b) asecond nucleotide sequence encoding a second polypeptide comprising atleast 200 amino acids, wherein the amino acid sequence of the secondpolypeptide and the amino acid sequence of SEQ ID NO:14 have at least85% identity based on the Clustal alignment method, (c) a thirdnucleotide sequence encoding a third polypeptide comprising at least 300amino acids, wherein the amino acid sequence of the third polypeptideand the amino acid sequence of SEQ ID NO:18 have at least 70% identitybased on the Clustal alignment method, or (d) the complement of thefirst, second, or third nucleotide sequence, wherein the complement andthe first, second, or third nucleotide sequence contain the same numberof nucleotides and are 100% complementary.
 2. The polynucleotide ofclaim 1, wherein the amino acid sequence of the first polypeptide andthe amino acid sequence of SEQ ID NO:4 or SEQ ID NO:10 have at least 80%identity based on the Clustal alignment method, and wherein the aminoacid sequence of the third polypeptide and the amino acid sequence ofSEQ ID NO:18 have at least 80% identity based on the Clustal alignmentmethod.
 3. The polynucleotide of claim 1, wherein the amino acidsequence of the first polypeptide and the amino acid sequence of SEQ IDNO:4 or SEQ ID NO:10 have at least 85% identity based on the Clustalalignment method, and wherein the amino acid sequence of the thirdpolypeptide and the amino acid sequence of SEQ ID NO:18 have at least85% identity based on the Clustal alignment method.
 4. Thepolynucleotide of claim 1, wherein the amino acid sequence of the firstpolypeptide and the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:10have at least 90% identity based on the Clustal alignment method,wherein the amino acid sequence of the second polypeptide and the aminoacid sequence of SEQ ID NO:14 have at least 90% identity based on theClustal alignment method, and wherein the amino acid sequence of thethird polypeptide and the amino acid sequence of SEQ ID NO:18 have atleast 90% identity based on the Clustal alignment method.
 5. Thepolynucleotide of claim 1, wherein the amino acid sequence of the firstpolypeptide and the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:10have at least 95% identity based on the Clustal alignment method,wherein the amino acid sequence of the second polypeptide and the aminoacid sequence of SEQ ID NO:14 have at least 95% identity based on theClustal alignment method, and wherein the amino acid sequence of thethird polypeptide and the amino acid sequence of SEQ ID NO:18 have atleast 95% identity based on the Clustal alignment method.
 6. Theisolated polynucleotide of claim 1, wherein the first polypeptidecomprises the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:10,wherein the second polypeptide comprises the amino acid sequence of SEQID NO:14, and wherein the third polypeptide comprises the amino acidsequence of SEQ ID NO:18.
 7. The isolated polynucleotide of claim 1,wherein the first nucleotide sequence comprises the nucleotide sequenceof SEQ ID NO:3 or SEQ ID NO:9, wherein the second nucleotide sequencecomprises the nucleotide sequence of SEQ ID NO:13, and wherein the thirdnucleotide sequence comprises the nucleotide sequence of SEQ ID NO:17.8. The isolated polynucleotide of claim 1, wherein the first, second,and third polypeptides are brittle-1 homologs.
 9. A chimeric genecomprising the polynucleotide of claim 1 operably linked to a regulatorysequence.
 10. A vector comprising the polynucleotide of claim
 1. 11. Anisolated polynucleotide fragment comprising a nucleotide sequencecomprised by the polynucleotide of claim 1, wherein the nucleotidesequence contains at least 30 nucleotides.
 12. The fragment of claim 11,wherein the nucleotide sequence contains at least 40 nucleotides. 13.The fragment of claim 11, wherein the nucleotide sequence contains atleast 60 nucleotides.
 14. An isolated polypeptide comprising: (a) afirst amino acid sequence comprising at least 200 amino acids, whereinthe first amino acid sequence and the amino acid sequence of SEQ ID NO:4or SEQ ID NO:10 have at least 70% identity based on the Clustalalignment method, (b) a second amino acid sequence comprising at least200 amino acids, wherein the second amino acid sequence and the aminoacid sequence of SEQ ID NO:14 have at least 85% identity based on theClustal alignment method, or (c) a third amino acid sequence comprisingat least 300 amino acids, wherein the third amino acid sequence and theamino acid sequence of SEQ ID NO:18 have at least 70% identity based onthe Clustal alignment method.
 15. The polypeptide of claim 14, whereinthe first amino acid sequence and the amino acid sequence of SEQ ID NO:4or SEQ ID NO:10 have at least 80% identity based on the Clustalalignment method, and wherein the third amino acid sequence and theamino acid sequence of SEQ ID NO:18 have at least 80% identity based onthe Clustal alignment method.
 16. The polypeptide of claim 14, whereinthe first amino acid sequence and the amino acid sequence of SEQ ID NO:4or SEQ ID NO:10 have at least 85% identity based on the Clustalalignment method, and wherein the third amino acid sequence and theamino acid sequence of SEQ ID NO:18 have at least 85% identity based onthe Clustal alignment method.
 17. The polypeptide of claim 14, whereinthe first amino acid sequence and the amino acid sequence of SEQ ID NO:4or SEQ ID NO:10 have at least 90% identity based on the Clustalalignment method, wherein the second amino acid sequence and the aminoacid sequence of SEQ ID NO:14 have at least 90% identity based on theClustal alignment method, and wherein the third amino acid sequence andthe amino acid sequence of SEQ ID NO:18 have at least 90% identity basedon the Clustal alignment method.
 18. The polypeptide of claim 14,wherein the first amino acid sequence and the amino acid sequence of SEQID NO:4 or SEQ ID NO:10 have at least 95% identity based on the Clustalalignment method, wherein the second amino acid sequence and the aminoacid sequence of SEQ ID NO:14 have at least 95% identity based on theClustal alignment method, and wherein the third amino acid sequence andthe amino acid sequence of SEQ ID NO:18 have at least 95% identity basedon the Clustal alignment method.
 19. The polypeptide of claim 14,wherein the first amino acid sequence comprises the amino acid sequenceof SEQ ID NO:4 or SEQ ID NO:10, wherein the second amino acid sequencecomprises the amino acid sequence of SEQ ID NO:14, and wherein the thirdamino acid sequence comprises the amino acid sequence of SEQ ID NO:18.20. The polypeptide of claim 14, wherein the polypeptide is a brittle-1homolog.
 21. A method for transforming a cell comprising introducing thepolynucleotide of claim 1 into a cell.
 22. A cell comprising thechimeric gene of claim
 9. 23. A method for producing a transgenic plantcomprising transforming a plant cell with the polynucleotide of claim 1and regenerating a plant from the transformed plant cell.
 24. A plantcomprising the chimeric gene of claim
 9. 25. A seed comprising thechimeric gene of claim 9.